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THE 






BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 



Editorial Board 






JOHN M. ANDERSON, Cornell University ROBERT K. JOSEPHSON, University of 

California. Irvine 
ARTHUR L. COLWIN, Queens College, New York 

F. H. RUDDLE, Yale University 
DONALD P. COSTELLO, University of 

North Carolina BERTA SCHARRER, Albert Einstein College 

of Medicine 
PHILIP B. DUNHAM, Syracuse University 

MELVIN SPIEGEL, Dartmouth College 
CATHERINE HENLEY, University of 

North Carolina STEPHEN A. WAINWRIGHT, Duke University 
MEREDITH L. JONES, Smithsonian Institution CARROLL M. WILLIAMS, Harvard University 

W. D. RUSSELL-HUNTER, Syracuse University 
Managing Editor 



VOLUME 143 

JULY TO DECEMBER, 1972 







Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE 8C LEMON STS. 

LANCASTER, PA. 



11 

THE BIOLOGICAL BULLETIN is issued six times a year at the 
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- 
sylvania. 

Subscriptions and similar matter should be addressed to The 
Biological Bulletin, Marine Biological Laboratory, Woods Hole, 
Massachusetts. Agent for Great Britain : Wheldon and Wesley, 
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London. 
W. C. 2. Single numbers, $5.00. Subscription per volume (three 
issues), $14.00. 

Communications relative to manuscripts should be sent to Dr. 
W. D. Russell-Hunter, Marine Biological Laboratory, Woods 
Hole, Massachusetts 02543 between May 23 and September 1, 
and to Dr. W. D. Russell-Hunter, P.O. Box 103, University 
Station, Syracuse, New York 13210, during the remainder of 
the year. 



Second-class postage paid at Lancaster, Pa. 



I \NCASTFR PRESS, INC., LANCASTER, PA. 




CONTENTS 

No. 1, AUGUST. l l >72 

Annual Report of the Marine Biological Laboratory 

ACHE, BARRY W. AND DEMOREST DAVENPORT 

The sensor\- basis of host recognition by symbiotic shrimps, genus 
Betacns 94 

FORWARD, RICHARD li., JR.. KENNETH W. HORCH AND TALBOT H. WATER- 
MAN 
Visual orientation at the water surface by the teleost Zenar chapter us. . . 112 

FRAENKEL, G., JAN ZDAREK AND P. SIVASUBRAMANIAN 

Hormonal factors in the CNS and hemolymph of pupariating fly larvae 
which accelerate puparium formation and tanning 127 

FUKE, M. T. AND T. SUGAI 

Studies on the naturally occurring hemagglutinin in the coelomic fluid 

of an ascidian 140 

HUGHES, G. M. AND I. E. GRAY 

Dimensions and ultrastructure of toadfish gills 150 

IVKER, FRANCES B. 

A hierarchy of histo-incompatibility in Hydractinia cchlnata 162 

LEVINTON, JEFFREY 

Spatial distribution of Xncnla [>ro.viina Say ( Protobranchia) : an experi- 
mental approach 17? 

MANTON. MARION, ANDREW KARR AND DAVID W. EHRENFELD 

Chernoreception in the Mugratory sea turtle, Clielonm uivdas 184 

McMuRRY, LAURA AND J. W. HASTINGS 

Orcadian rhythms: mechanism of luciferase activity changes in 
(t'onyaitla.r 196 

REEVE, M. R. AND M. A. WALTER 

Observations and experiments on methods of fertilization in the chae- 
tognath Sci(/itta hispida 207 

SCHAUER, RUTH YANSTORY 

Excystation of the apostomatous ciliate. H\alo[>liysa chaltoni, without 
metamorphosis 21? 

SKINNER, DOROTHY M. AND DALE E. GRAHAM 

Loss of limbs as a stimulus to ecdysis in Brachyura (true crabs ) 222 

SMITH, RALPH I. AND PAUL P. RUDY 

"Water-exchange in the crab Hemigrapsiis tindns measured by use of 
deuterium and tritium oxides as tracers 234 

STIFFLER, DANIEL F. AND AUSTIN \Y. PRITCHAKD 

A comparison of In situ and in rltro responses of crustacean hearts to 
hypoxia 

YOUNG, PAUL G.. A. DOROTHY YOUNG AND ARTHUR M. ZIMMERMAN 

Action of hydrostatic pressure on sea urchin cilia 2?0 



in 



iv CONTENTS 

X<>. 2. OCTOBER, 1972 

BELL, \YAYNE AND RALPH MITCHELL 

Chemotactic and growth responses of marine bacteria to algal extra- 
cellular products 265 

BROWN, STEPHEN C, JOHN B. BDZIL. AND HARRY I,. FRISCII 

Responses of Chaetopterns variopedatus to osmotic stress, with a dis- 
cussion of the mechanism of isoosmotic volume-regulation 278 

BRUMMETT, ANNA RUTH AND \\"INONA 15. YERNBEKC 

Oxygen consumption in anterior versus posterior embryonic shield of 
Finufiilus heteroclitns 296 

BURKY, ALBERT J., J. PACHECO, AND EUC;ENIA PEREYKA 

Temperature, water, and respiratory regimes of an amphibious snail. 
1'oinacea iirceus ( Mullev I from the Yenexnelan savannah ^04 

CARRIKER, MELBOURNE R., I'IIILIP PKRSOX. RICHARD LIBP.IX, AND DIRK 
VAN ZANDT 

Regeneration of the proboscis of muricid gastropods after amputation, 
with emphasis on the radula and cartilages ^17 

DUNLAP, DONALD G. 

Latitudinal effects on metabolic rates in the cricket frog. .Icris crepitans: 
acutely measured rates in summer frogs ^32 

HEIDC.ER, PAUL M., |R.. ROBERT G. SUMMERS, AND IAMF.S A. MILLER. !R. 

llvperbaric oxygen and ( mbryonic develo])ment in .Irhacia pitnctitlata . . 344 

HINSCH, GERTRUDE \Y. 

Some factors controlling reproduction in the spider crab. Libinia cinari/i- 
nuta 358 

JUNCREIS, ARTHUR M. AXD(J. R. \YYATT 

Sugar release and penetration in insect fat bod\ : relations to regulation 

of haemolymph trehalosc- in developing stages of Hydlophm-a cccropia . . 367 

I .AI.LT, CAROL M. 

Food and feeding of Paedoclione doliiformis Danforth, a neotenous 
gymnosomatous pteropod 392 

AlARTINSEN, DAVID L. AND D()NALD |. KlMELDORE 

The prompt detection of ionizing radiations by carpenter ants 403 

NOZAWA, K., D. L. TAYLOR, AND L. PROYASOLI 

Respiration aiul photosynthesis in Convoluta roscoffensis ( iraff, infected 
with Yarious symbionts 420 

OZAWA, EIJIRO 

The role of calcium ion in avian nnogenesis in vitro 431 

SPAULDING, JAMES G. 

The life cycle of PcacJiia (juinqncca^itata. an anemone parasitic on 
medusae during its larval development 44( ) 

\i)>tracts of papers presented at the Marine Biological Laboratory 4?4 



CONTENTS v 

No. 3. DECEMBER, 1972 

CASSIDY, JOSEPH D., O. P. AND ROBERT C. KING 

Ovarian development in Habrobracon jiu/Unulis ( Ashmead ) ( Hy- 
menoptera: Braconiclae ) . 1. The origin and differentiation of the 
oocyte-nurse cell complex 483 

CRENSHAW, MILES A. 

The inorganic composition of molluscan extrapallial fluid 506 

DOWSE, H. BURGESS AND JOHN D. PALMER 

The chronomutagenic effect of deuterium oxide 011 the period and en- 
traimnent of a biological rhythm ^1^ 

JOHNSON, JOAN HEWLETT AND ROBERT C. KING 

Studies on Fes, a mutation affecting cystocyte cytokinesis, in Drosophila 
inelanogastcr 525 

KENNEY, DIANNE M., FRANK A. BELAMARICH AND DAVID SHEPRO 

Aggregation of horseshoe crab ( Limn/us polypheinits) amebocytes and 
reversible inhibition of aggregation by EDTA 548 

KLAPOW, L. A. 

Fortnightly molting and reproductive cycles in tlie sand-beach isopod, 
Excirolana chill oni 568 

PAPARO, ANTHONY 

Innervation of the lateral cilia in the mussel. Myfilns etlnlis 1 592 

PAPPAS, PETER \\'. AND CLARK IV READ 

Inactivation of <*- and /S-chymotrypsin by intact Hymenolepis diminuta 
(Cestoda) 605 

RICE, NOLAN E. AND \Y. ALLAN POWELL 

Observations on three species of jellyfishes from Chesapeake Bay with 
special reference to their toxins. 1 1. Cynnca capillahi 617 

RUSSELL-HUNTER, W. D., MARTYN L. ApLEY AND R. DofGLAS 1 ll'NTER 

Early life-history of Mela in pus and the significance of semilunar syn- 
chrony 623 

SASSAMAN, CLAY AND CHARLOTTE P. MANGUM 

Adaptations to environmental oxygen levels in infaunal and epifaunal sea 
anemones 657 

SCOTT, DANA M. AND C. \V. MAJOR 

The effect of copper (IT) on survival, respiration, and lu-art rate in the 
common blue mussel. Mytilns edit I is 67 ( 

SHORE, RICHARD E. 

Axial filament of silicious sponge spicules, its organic components and 
synthesis 689 

ZEUTHEN, ERIK AND KIRSTEN HAMBURGER 

Mitotic cycles in oxygen uptake and carbon dioxide output in the cleaving 
frog egg 699 



Vol. 143, No. 1 August, 1972 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



THE MARINE BIOLOGICAL LABORATORY 
SEVENTY-FOURTH REPORT, FOR THE YEAR 1971 EIGHTY-FOURTH YEAR 

I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST, 1971) 1 

II. ACT OF INCORPORATION 4 

III. BYLAWS OF THE CORPORATION 5 

IV. REPORT OF THE DIRECTOR 7 

Addenda : 

1. Memorials 13 

2. The Staff. .... 20 

3. Investigators, Fellowships, and Students 33 

4. Fellows and Scholarships 48 

5. Training Programs 48 

6. Tabular View of Attendance, 1967 1971 51 

7. Institutions Represented 51 

8. Friday Evening Lectures 53 

9. Tuesday Evening Seminars 54 

10. Members of the Corporation 55 

V. REPORT OF THE LIBRARIAN .... 83 

VI. REPORT OF THE TREASURER. 84 



I. TRUSTEES 
Including Action of 1971 Annual Meeting 

DENIS M. ROBINSON, Chairman of the Board of Trustees, 19 Orlando Avenue, Arlington, 

Massachusetts 02174 
GERARD SWOPE, JR., Honorary Chairman of the Board of Trustees, Croton-on-Hudson, 

New York, New York 10520 

ALEXANDER T. DAIGNAULT, Treasurer, 7 Hanover Square, New York, New York 10005 
JAMES D. EBERT, Director and President of the Corporation, Director, Department of 

Embryology, Carnegie Institution 
DAVID SHEPRO, Clerk of the Corporation, Boston University, Boston 

1 

Copyright 1972, by the Marine Biological Laboratory 
Library of Congress Card No. A38-518 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

EMERITI 

WILLIAM R. ARMSTRONG, Falmouth, Massachusetts 

PHILLIP B. ARMSTRONG, State University of New York, College of Medicine, Syracuse 

DETLEV W. BRONK, The Rockefeller University 

C. LALOR BURDICK, The Lalor Foundation 

E. G. BUTLER, Princeton University 

C. LLOYD CLAFF, Brockton, Massachusetts 

KENNETH S. COLE, National Institutes of Health 

PAUL S. GALTSOFF, Woods Hole, Massachusetts 

RUDOLF T. KEMPTON, Vassar College 

DOUGLAS MARSLAND, Marine Biological Laboratory 

CHARLES W. METZ, Woods Hole, Massachusetts 

CHARLES PACKARD, Woods Hole, Massachusetts 

HAROLD H. PLOUGH, Amherst, Massachusetts 

A. C. REDFIELD, Woods Hole, Massachusetts 

CARL C. SPEIDEL, University of Virginia 

A. H. STURTEVANT, California Institute of Technology 
ALBERT SZENT-GYORGYI, Marine Biological Laboratory 
W. RANDOLPH TAYLOR, University of Michigan 

B. H. WILLIER, The Johns Hopkins University 

CLASS OF 1975 

WILLIAM J. ADELMAN, National Institutes of Health 

FRANCIS D. CARLSON, The Johns Hopkins University 

LAURA H. COLWIN, Queens College 

SEARS CROWELL, Indiana University 

CATHERINE HENLEY, University of North Carolina 

SAMUEL LENHER, Wilmington, Delaware 

JOHN W. MOORE, Duke University Medical Center 

W. D. RUSSELL-HUNTER, Syracuse University 

WALTER S. VINCENT, University of Delaware 

CLASS OF 1974 

ROBERT D. ALLEN, State University of New York at Albany 

MICHAEL V. L. BENNETT, Albert Einstein College of Medicine 

JOHN E. DOWLING, Johns Hopkins University 

HARLYN O. HALVORSON, University of Wisconsin 

J. WOODLAND HASTINGS, Harvard University 

JAMES W. LASH, University of Pennsylvania 

RICHARD S. MORSE, \Vellesley, Massachusetts 

CLARK P. READ, Rice University 

H. BURR STEINBACH, Woods Hole Oceanographic Institution 

CLASS OF 1973 

JAMES CASE, University of California, Santa Barbara 

ARTHUR L. COLWIN, Queens College 

WILLIAM T. GOLDEN, New York, New York 

GEORGE G. HOLZ, JR., State University of New York, Upstate Medical Center, Syracuse 

SHINYA INOUE, University of Pennsylvania 

CHARLES B. METZ, University of Miami 



TRUSTEES 

GEORGE T. SCOTT, Oberlin College 

MALCOLM S. STEINBERG, Princeton University 

CLASS OF 1972 

JOHN B. BUCK, National Institutes of Health 

ANTHONY C. CLEMENT, Emory University 

DONALD P. COSTELLO, University of North Carolina 

GEORGE H. A. CLOWES, JR., Harvard Medical School 

TERU HAYASHI, Illinois Institute of Technology 

ALBERTO MONROY, University of Palermo, Italy 

JOHN W. SAUNDERS, JR., State University of New York at Albany 

HOWARD A. SCHNEIDERMAN, University of California, Irvine 

ANDREW SZENT-GYORGYI, Brandeis University 



STANDING COMMITTEES 

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES 

DENNIS M. ROBINSON, ex officio JOHN E. DOWLING, 1973 

ALEXANDER T. DAIGNAULT, ex officio HARLYN O. HALVORSON, 1973 

JAMES D. EBERT, ex officio JOHN B. BUCK, 1972 

J. WOODLAND HASTINGS, 1974 WALTER S. VINCENT, 1972 
JAMES W. LASH, 1974 

LIBRARY COMMITTEE 

CATHERINE HENLEY, Chairman THOMAS J. SCHOPF 

GARLAND E. ALLEN RUBERT ANDERSON 

FRED GRASSLE NORMAN B. RUSHFORTH 

IVAN VALIELA T. FERRIS WEBSTER 
BRUCE WARREN 

RESEARCH SERVICES COMMITTEE 

H. A. DEPHILLIPS, Chairman ROBERT V. RICE 

W. J. ADELMAN M. S. STEINBERG 

HERMAN BOSCH ANDREW SZENT-GYORGYI 

ANTHONY LIUZZI DAVID VPHANTIS 

SUPPLY DEPARTMENT COMMITTEE 

MILTON FINGERMAN, Chairman DAVID GRANT 

LAWRENCE COHEN RALPH HINEGARDNER 

SEARS CROWELL W. D. RUSSELL-HUNTER 

FRANK FISHER \VILLIAM STEWART 

INSTRUCTION COMMITTEE 

PHILIP B. DUNHAM, Chairman HUGH HUXLEY 

F. D. CARLSON JOHN M. TEAL 

MAURICE Fox J. R. WHITTAKER 
AUDREY HASCHEMEYER 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

BUILDINGS AND GROUNDS COMMITTEE 

PHILIPP STRITTMATTER, Chair man GERTRUDE HINSCH 

EVERETT ANDERSON LEONARD NELSON 

D. EUGENE COPELAND LEON P. WEISS 

JAMES GREEN CHARLES WYTTENBACH 

RADIATION COMMITTEE 

GEORGE T. REYNOLDS, Chairman LASZLO LORAND 

J. R. COLLIER JAMES MORIN 

DANIEL GROSCH R. C. RUSTAD 

RESEARCH SPACE COMMITTEE 

J. \V. HASTINGS, Chairman H. JANNASCH 

J. F. CASE J. \V. LASH 

A. L. COLWIN \\'. S. VINCENT 
T. H. GOLDSMITH 

COMMITTEE FOR THE NOMINATION OF OFFICERS 

WALTER S. VINCENT JOHN B. BUCK 

HARLYN O. HALVORSOX JOHN E. DOWLING 

JAMES W. LASH J. WOODLAND HASTINGS 

FOOD SERVICE COMMITTEE 

JOHN ARNOLD, Chairman G. H. COUSINEAU 

FR. J. D. CASSIDY A. FARMANFARMAIAN 

S. J. COOPERSTEIN RlTA GUTTMAN 

COMPUTER SERVICES COMMITTEE 

JOHN W. MOORE, Chairman MELVIN ROSENFELD, JR. 

ARNOLD LAZAROW NORMAN B. RUSHFORTH 

C. LEVINTHAL 



II. ACT OF CORPORATION 

No. 3170 

COMMONWEALTH OF MASSACHUSETTS 

Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. 
Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, 
William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves 
with the intention of forming a Corporation under the name of the Marine Biological 
Laboratory, for the purpose of establishing and maintaining a laboratory or station for 
scientific study and investigation, and a school for instruction in biology and natural his- 
tory, and have complied with the provisions of the statutes of this Commonwealth in 
such case made and provided, as appears from the certificate of the President, Treasurer, 
and Trustees of said Corporation, duly approved by the Commissioner of Corporations, 
and recorded in this office; 

Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- 
setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi- 



ACT OF INCORPORATION 5 

ner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, 
their associates and successors, are legally organized and established as, and are hereby 
made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB- 
ORATORY, with the powers, rights, and privileges, and subject to the limitations, 
duties, and restrictions, which by law appertain thereto. 

Witness my official signature hereunto subscribed, and the seal of the Commonwealth 
of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord 
One Thousand Eight Hundred and Eighty-Eight. 

[SEAL] HENRY B. PIERCE, 

Secretary of the Commonwealth 



III. BYLAWS OF THE CORPORATION OF THE MARINE 
BIOLOGICAL LABORATORY 

(Revised February 11, 1972) 

I. The members of the Corporation shall consist of persons elected by the Board 
of Trustees. 

II. The officers of the Corporation shall consist of a Chairman of the Board of 
Trustees, President, Director, Treasurer and Clerk. 

III. The Annual Meeting of the members shall be held on the Friday following the 
second Tuesday in August in each year at the Laboratory in Woods Hole, Massachusetts, 
at 9:30 A.M., and at such meeting the members shall choose by ballot a Treasurer and a 
Clerk to serve one year, and nine Trustees to serve four years, and shall transact such 
other business as may properly come before the meeting. Special meetings of the 
members may be called by the Trustees to be held at such time and place as may be 
designated. 

IV. Twenty-five members shall constitute a quorum at any meeting. 

V. Any member in good standing may vote at any meeting, either in person or by 
proxy duly executed. 

VI. Inasmuch as the time and place of the Annual Meeting of members are fixed by 
these bylaws, no notice of the Annual Meeting need be given. Notice of any special 
meeting of members, however, shall be given by the Clerk by mailing notice of the time 
and place and purpose of such meeting, at least (15) days before such meeting, to each 
member at his or her address as shown on the records of the Corporation. 

VII. The Annual Meeting of the Trustees shall be held promptly after the Annual 
Meeting of the Corporation at the Laboratory in W r oods Hole, Massachusetts. Special 
meetings of the Trustees shall be called by the Chairman, the President, or by any seven 
Trustees, to be held at such time and place as may be designated, and the Secretary 
shall give notice thereof by written or printed notice, mailed to each Trustee at his 
address as shown on the records of the Corporation, at least one (1) week before the 
meeting. At such special meeting only matters stated in the notice shall be considered. 
Seven Trustees of those eligible to vote shall constitute a quorum for the transaction 
of business at any meeting. 

VIII. There shall be three groups of Trustees: 

(A) Thirty-six Trustees chosen by the Corporation, divided into four classes, each 
to serve four years. After having served two consecutive terms of four years each, 
Trustees are ineligible for re-election until a year has elapsed. 



6 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

(B) Trustees ex officio, who shall be the Chairman, the President, the Director, 
the Treasurer, and the Clerk. 

(C) Trustees Emeriti, who shall be elected from present or former Trustees by the 
Corporation. Any member of the Corporation in good standing who has attained the 
age of seventy years, or has attained the age of sixty-five and has retired from his home 
institution, and who has served a full elected term as a regular Trustee, shall be desig- 
nated Trustee Emeritus for life at the next annual meeting provided he signifies his 
wish to serve the Laboratory in that capacity. Any regular trustee who qualifies for 
emeritus status shall continue to serve as Trustee until the next Annual Meeting 
whereupon his office as regular Trustee shall become vacant and be filled by election by 
the Corporation. The Trustees ex officio and Emeriti shall have all the rights of the 
Trustees, except that Trustees Emeriti shall not have the right to vote. 

The Trustees and officers shall hold their respective offices until their successors are 
chosen and have qualified in their stead. 

IX. The Trustees shall have the control and management of the affairs of the Cor- 
poration. They shall elect a Chairman of the Board of Trustees who shall be elected 
annually and shall serve until his successor is selected and qualified and who shall also 
preside at meetings of the Corporation. They shall elect a President of the Corporation 
who shall also be the Vice Chairman of the Board of Trustees and Vice Chairman of 
meetings of the Corporation, and who shall be elected annually and shall serve until his 
successor is selected and qualified. They shall appoint a Director of the Laboratory for 
a term not to exceed five years, provided the term shall not exceed one year if the candi- 
date has attained the age of 65 years prior to the date of the appointment. They may 
choose such other officers and agents as they may think best. They may fix the com- 
pensation and define the duties of all the officers and agents; and may remove them, or 
any of them except those chosen by the members, at any time. They may fill vacancies 
occurring in any manner in their own number or in any of the officers. The Board of 
Trustees shall have the power to choose an Executive Committee from their own num- 
ber, and to delegate to such Committee such of their own powers as they may deem 
expedient. They shall from time to time elect members to the Corporation upon such 
terms and conditions as they may think best. 

X. The Associates of the Marine Biological Laboratory shall be an unincorporated 
group of persons (including associations and corporations) interested in the Laboratory 
and shall be organized and operated under the general supervision and authority of the 
Trustees. 

XI. The consent of every Trustee shall be necessary to dissolution of the Marine 
Biological Laboratory. In case of dissolution, the property shall be disposed of in such 
manner and upon such terms as shall be determined by the affirmative vote of two-thirds 
of the Board of Trustees. 

XII. The account of the Treasurer shall be audited annually by a certified public 
accountant. 

XIII. These bylaws may be altered at any meeting of the Trustees, provided that 
the notice of such meeting shall state that an alteration of the bylaws will be acted upon. 

RESOLUTIONS ADOPTED AT TRUSTEES' MEETINGS 
EXECUTIVE COMMITTEE 

I. RESOLVED: 

(A) The Executive Committee is hereby designated to consist of not more than ten 
members, including the ex officio members (Chairman of the Board of Trustees, Presi- 



REPORT OF THE DIRECTOR 7 

dent, Director and Treasurer) ; and six additional Trustees, two of whom shall be elected 
by the Board of Trustees each year, to serve for a three-year term. (August 11, 1967). 

(B) The Chairman of the Board of Trustees shall act as Chairman of the Executive 
Committee, and the President as Vice President. A majority of the members of the 
Executive Committee shall constitute a quorum and a majority of those present at any 
properly held meeting shall determine its action. It shall meet at such times and places 
and upon such notice and appoint such sub-committees as the Committee shall deter- 
mine. (August 12, 1966). 

(C) The Executive Committee shall have and may exercise all the powers of the 
Board during the intervals between meetings of the Board of Trustees except those 
powers specifically withheld from time to time by the Board or by law. (August 16, 
1963). 

(D) The Executive Committee shall keep appropriate minutes of its meetings, and 
its action shall be reported to the Board of Trustees. (August 16, 1963). 

II. RESOLVED: 

The elected members of the Executive Committee be constituted as a standing "Com- 
mittee for the Nominations of Officers," responsible for making nominations, at each 
Annual Meeting of the Corporation, and of the Board of Trustees, for candidates to fill 
each office as the respective terms of office expire (Chairman of the Board, President, 
Director, Treasurer, and Clerk). (August 16, 1963). 



iv. REPORT OF THE DIRECTOR 

To: THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY 

Gentlemen : 

This report to the Trustees of the Marine Biological Laboratory is made at a time 
when past programs are being reviewed, when the Laboratory's objectives and the 
means earlier employed in seeking them are being restudied, and when the basic plans 
by which the Laboratory's scientific work is carried on are being reformulated. 

The reassessment of objectives and procedures should be a continuing element in the 
conduct of any scientific institution ; it has been such throughout the history of the 
Laboratory. But as Vannevar Bush wrote about another institution at a watershed, 
only rarely indeed do circumstances so shape themselves that the re-evaluation becomes 
itself a primary undertaking extending practically throughout the Laboratory. 

This rigorous evaluation of the several programs by which, taken as a whole, the 
Laboratory carries out its mandate, is now in its second year. Thus far three general 
observations have emerged. First, we rightly take from this study renewed confidence 
in the central philosophy of the Laboratory. Secondly, it is clear that our scientists 
respect and draw heavily upon the resourcefulness of the Laboratory's able and loyal 
administrative and technical staff. Thirdly, our traditional organization is out of step 
with the realities of science support today. 

The first two conclusions are reassuring. It is the third observation that gives cause 
for continuing concern. Our pattern of organization which in the past, and to a large 
extent, even today, has proved to be highly effective in providing opportunities for the 
productive investigator and promising student, in making available resources to leaders 
of proved skill in limning the unknown, may no longer provide adequate mechanisms for 
maintaining the financial viability of the Laboratory. 

From the very beginning the Laboratory has sought the exceptional man and the 
exceptional program. ITntil recently our ability to compete for federal grants for 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

graduate education and tor institutional support made it possible for the Laboratory, 
at least to a very large extent, to march to its own drummer. Now the inability of the 
responsible federal agencies to respond to our proposals in these categories and the 
gradual restriction of their mandate to contracts and grants for circumscribed projects 
make it essential that we educe new ways of attaining our objectives in the face of ever- 
changing and ever-more restrictive federal guidelines. 

Is our organization prepared to respond rapidly to changes required as new programs 
unfold? If not, what improvements should be made (and at what cost)? Which 
programs are most urgent? Which are likely to be most productive? Which have 
served their purpose? These are the principal questions to which the Officers and 
Executive Committee have addressed themselves during the year. In this review they 
have worked closely with four committees. 

The mission of the Finance Committee is to reinforce the Executive Committee by 
providing a thorough assessment of the budget and the measures required to improve 
our financial situation. Its members are Robert Allen, Alexander T. Daignault (Chair- 
man), William T. Golden, Samuel Lenher and John W. Moore. 

The Committee on Winter Operations, chaired by George Holz, was asked to assess the 
current performance of the Laboratory in winter operations and to recommend changes 
required if and when those operations are increased. Its roster included George H. 
Clowes, Sears Crowell, A. L. Gorman, Clifford Harding, Robert Josephson, Robert 
Kahler, Hans Laufer, Yoram Palti and John Valois. 

Upon Edgar Zwilling's untimely death, Eric Ball assumed the chairmanship of the 
Salary Review Panel. Despite his own illness and the sensitive nature of the assignment, 
he and his colleagues (John Arnold, M. R. Carriker and Catherine Henley) submitted a 
reasoned, thoughtful report which has already led to significant changes. The Panel 
itself has been discharged but arising out of it are two new bodies, a continuing Com- 
mittee on Employee Relations and an ad hoc Retirement Committee. The former group 
includes three employees, Edward Bender, Robert Gunning and John Valois (Chairman) 
and three members of the Corporation (Lucena Earth, Catherine Henley, Ivan Valiela, 
serving one, two and three year terms, respectively). As vacancies occur by rotation, 
employees will elect their own representatives; the Corporation representatives will be 
appointed by the Director. 

John Arnold (Chairman), Gertrude Hinsch and J. W. Lash constitute the Retirement 
Committee, which has been asked to recommend improvements in the Laboratory's 
pension program which might be put into force by January 1, 1973. 

This year, as last, I have placed emphasis on contributions arising from interactions 
of many Corporation members and employees. From such cross-linkages we may ex- 
pect added effectiveness, providing their recommendations are regarded, not as rigid 
prescriptions, but as signals to the Trustees who are the arbiters of our long-range policy. 

It is against this background that I shall summarize the principal actions and de- 
cisions of the past year. 

The Laboratory as a year-roitnd center 

Although our proposal submitted to the National Science Foundation on May 12, 1971 
has not been officially declined at this writing, we have been informed that regulations 
of the Office of Management and Budget currently in force prevent the Foundation 
from providing institutional support or from considering the Laboratory as a modified 
national laboratory, as discussed by our Executive Committee with members of the 
Foundation's staff. \Ve have been advised that the Foundation is prepared to evaluate 
research proposals from individuals and teams, but that it cannot provide the "primer" 
funds called for in our proposal to equip and staff the Laboratory for year-round re- 
search. One of the actions taken by our Trustees at their meeting on February 11, 1972 



REPORT OF THE DIRECTOR 9 

has an important bearing on the question. In approving the Executive Committee's 
resolution that a Deputy Director be appointed to be in residence throughout the year, 
the Trustees expressed their confidence that, providing someone with the requisite 
breadth of vision in research and qualities of leadership can be found, a research nucleus 
can be established that will ultimately act as a focus for the development of a series of 
related programs. The identification of such a "chief scientist in residence," working 
closely with the Director and General Manager should permit such a division of labor 
and distribution of responsibilities as to insure a more effective overview of the Labora- 
tory's needs. 

At the same time, we are attempting to attract small groups of highly competent 
investigators to the Laboratory. By emphasizing groups, we do not mean to cate- 
gorically exclude the lone independent scientist. However, we do wish to stress in 
year-round programs, as we do in the summer, the advantages of exchange, of combining 
leaders of proved acumen and promising younger scientists, of balancing and combining 
the knowledge of several investigators. 

In addition, more than in the past, we shall try to carry on a continuing review of our 
year-round research. Some studies carried on for considerable periods in relative isola- 
tion appear to have reached the point where they should be carried forward by other 
agencies in locations where they can profit by more frequent peer review. 

It may appear paradoxical that while attempting to strengthen year-round research 
(including environmental biology), we have announced the termination of the System- 
atics-Ecology Program at the close of its first decade. The announcement was not made 
without regret, for not only was the concept of the Program as initiated by Philip 
Armstrong and Arthur Parpart bold and, in its day, forward-looking, but especially in 
its formative years the Program contributed significantly to the Laboratory's vitality. 
However, during the past few years its financial fortunes have steadily declined to the 
point at which no other decision appeared possible. I shall not enumerate the Program's 
many contributions, which its Director, M. R. Carriker will treat in his final report, 
nor shall I attempt to educe the reasons for its failure to attract funds, except to observe 
that during the period when the support of systematics was waning and of environmental 
biology waxing full, the Program's emphasis and strength tended to lie more on the side 
of systematics. It may also be true that, as one reviewer put it, "Systematics is scarcely 
more allied to ecology than to developmental biology." 

Winter teaching has been limited almost entirely to the Boston University Marine Pro- 
gram. During the second semester, 1971-1972, four staff members were in residence, the 
Director, Arthur Humes, William Stewart and Ivan Valiela being joined for the term 
by Stephan Golubic, teaching Marine Microphytes. Fourteen graduate students and 
four seniors participated for all or part of the year. At least 1 20 inquiries about graduate 
study have been received for the year beginning in September 1972. 

Beginning in January 1972. the first two undergraduate students of the Consortium 
of Northern New England arrived for the second semester. Five colleges and uni- 
versities offered formal courses at the Laboratory in the fall and spring, 1971-1972: 
Davidson College, Drew L T niversity, Oakland (Michigan) University, Temple University 
and University of Michigan. Three institutions conducted special programs for their 
students: Massachusetts Institute of Technology, LIniversity of Notre Dame, and 
Woods Hole Oceanographic Institution. 

The year also found a number of pre-college students using the Laboratory's facilities, 
space having been made available to the Falmouth school system for students in ad- 
vanced biology and in a marine sciences program. 

The summer courses: an agonizing reappraisal is necessary 

The quality of our summer offerings has rarely, if ever, been higher. At the close of 
the summer of 1971 we bid adieu to three Instructors-in-charge, James F. Case (Experi- 



10 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

mental Invertebrate Zoology). Andrew Szent-Gyorgyi (Physiology) and Malcolm 
Steinberg (Embryology), all of whom served responsibly and with distinction as leaders 
of innovative courses. In their places starting in 1972 are Robert Josephson, John 
Cebra and Eric Davidson. Of the three, only Josephson, now at the University of 
California, is a member of the Corporation, known personally to most readers of these 
Reports. Cebra, a distinguished immunochemist-cell biologist, Professor of Biology at 
Johns Hopkins, is a newcomer; while Davidson, who, like Cebra, was trained at Rocke- 
feller University, and is now a faculty member at California Institute of Technology, 
will be starting his second summer at the Laboratory. 

New leaders have also come forward to direct the Research Training Program in 
Excitable Membrane Biophysics and Physiology, David E. Goldman of Woman's 
Medical College of Pennsylvania and John W. Moore of Duke University having suc- 
ceeded W. J. Adelman who had guided the program since its inception. 

Having remarked upon the quality of our courses and some of their leaders, it seems 
incongruous to report that nearly half of the Laboratory's nine courses and training 
programs are operating in 1972 without sufficient funds. 

Four programs have federal funds. The Embryology course and the Research Train- 
ing Program in Fertilization and Gamete Physiology are supported by grants from the 
National Institute of Child Health and Human Development; the Physiology course 
continues to draw its funds from the National Institute of General Medical Sciences, 
and our program in the neural sciences for minority ethnic groups, Frontiers in Research 
and Teaching, is assured of three years' support from the National Institute of Neuro- 
logical Diseases and Stroke. 

A generous gift from Dr. Ernest B. Wright, supplemented by a grant from the 
Alfred P. Sloan Foundation, has permitted the continuation of the Program in Excitable 
Membrane Biophysics and Physiology. Neurobiology can be continued thanks to 
another gift from the Grass Foundation which has played so vital a role in training in 
the neurosciences at the Laboratory. However, it is essential that major support be 
found from other sources. The National Science Foundation has denied support to 
Ecology, Experimental Invertebrate Zoology and Experimental Marine Botany. For- 
tunately the Research Corporation has made a special award in support of an innovative 
program of faculty-student research in experimental marine ecology. Zoology and 
Botany will be offered in 1972, with the Laboratory providing minimal essential funding. 

Our proposals were declined because recommendations for their support would be 
contrary to the National Science Foundation's policy not to support teaching with 
research funds. Although there are large components of research in our proposals, 
with at least half the time of both staff and students being devoted to research, there is 
no denying that training is a primary objective. 

There is, in my judgment, a continuing need for such cousres in national centers like 
the Laboratory; courses like these are vital for maintaining the depth and robustness 
of science throughout the nation. The current policy at the National Science Founda- 
tion (which is, I believe, inimical to the Foundation's charge) will result in the well 
being of our science being left in the care of a community of aging scientists, with an 
attendant deterioration in innovation. The Foundation's policy denies the need for 
renewal. 

I am not alone in objecting to this short-sighted policy ; nonetheless I believe we must 
assume that it will not be reversed in the immediate future. Hence the heading of this 
section: an agonizing reappraisal. 

All of the facts are not yet in. The fundamental question is this: How will the com- 
plete lack of funds for student stipends, travel and tuition affect both the quality and 
numbers of students enrolled in these courses? The Laboratory cannot continue to 
fund these courses, even at a minimal level. They must be "self-supporting; here I 



REPORT OF THE DIRECTOR 11 

use "self" to mean that they must attract funds from a source other than the Laboratory 
itself. Even if we assume that the courses would be fully subscribed, with students 
(or their colleges and universities) willing to pay their fees, they would not be self- 
supporting at present levels of tuition, which are already high. 

In the weeks immediately ahead, the Officers and Trustees, the Instruction Com- 
mittee and Instructors-in-charge will have to find a solution. Some of the possibilities 
that have already emerged are the following: (1) The abolition of courses as they are 
presently constituted, with the development of summer research "program-projects" 
involving graduate students as research participants. It is a moot question whether 
such an approach could be funded. (2) A change in the character and structuring of 
the courses (possibly combining two or more), with a substantial reduction in the number 
of instructors; in short if sufficient paying students can be recruited to operate one 
or more courses on a break-even basis. (3) An even more drastic change in the character 
of our offerings for example we might offer refresher courses to college or high school 
teachers. 

At the risk of biasing future discussions, I would emphasize that I lean away from 
any solution in which we opt for mere survival a course for the sake of having a course. 
We have been pathfinders in graduate research training ; we must maintain our resiliency 
and find new ways of remaining in the vanguard. 

Support of the CAP'X BILL 

The National Science Foundation has approved our request for $15,000 per year for 
three years, beginning in 1972, for our project, "Support of a charter vessel for collecting 
materials for research in neurobiology." 

This grant will provide about half the cost of chartering the CAP'N BILL, the remainder 
to be obtained as in 1971, through a surcharge imposed on investigators using the ship's 
services. However, the grant will insure that students and younger independent in- 
vestigators who lack grant funds to cover such charges may have full access to squid and 
other animals provided by the CAP'N BILL. 

.1 nnual giving 

In the summer of 1971 the Laboratory initiated its Annual Giving Campaign, pat- 
terned after the annual "rollcalls" of colleges and universities, and successful fund- 
raising campaigns at our sister institutions, for example the Jackson Laboratory and 
the Cold Spring Harbor Laboratory. The idea did not meet with complete approval. 
At least a few members of the Corporation registered their concern that to put it as 
bluntly as one critic did an investigator might "buy his way into the Laboratory." 
Nevertheless, most of our members realized that it is a fact of life that we all inde- 
pendent laboratories are more dependent than ever on the help received from our 
local neighbors, our scientific alumni, foundations, and most importantly, our own 
members. \Ye must assign the Laboratory a high priority in our own giving. 

In the first year of the program, 177 Corporation members contributed and pledged 
(through May 18, 1972) $76,908. The Campaign will be renewed in October, 1972, 
and each October thereafter. Hopefully we will continue to merit the backing of those 
who have contributed in the past as well as of many new friends. 

MBL Award- 
In the summer of 1971, a generous gift from an anonymous donor enabled the Labora- 
tory to establish a prize to be known as the MBL Award, to be given for a noteworthy 
paper presented annually at the General Scientific Meetings. The monetary value of 
each award will be $100. 



12 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

It is a condition of the award that to be considered eligible the work must have been 
carried out at the Laboratory. Ordinarily the presentation at the General Scientific 
Meetings will have been the first public presentation (apart from discussions at depart- 
mental seminars and the like). Although some preference will be given to junior in- 
vestigators, it is recognized that occasionally established scientists have new ideas too. 
Accordingly no nominee will be excluded on the grounds of age. 

Judging will be based not only on the material presented at the Meeting but on a 
manuscript to be submitted by the nominee. Following the Meetings the chairmen of 
sessions, or any other listener, may submit in writing a nomination to the Director. 
Nominees will then be asked whether in their opinion their work meets the established 
criteria. If the paper is eligible, the nominee will then submit to the Director within 
four months following the meeting a description of the principal findings not to exceed 
1500 words, together with any other supporting data he sees fit to include. 

The Committee of Judges had a difficult time in selecting the first winner, so difficult 
in fact that it has decided upon a dual award : to J. E. Lisman and J. E. Brown for their 
paper, "Effect of intracellular pressure injection of Ca + ^-EGTA buffers into Limiilns 
ventral photoreceptors" and J. R. \\ hittaker, whose contribution was entitled 
"Differentiation without cleavage in an ascidian egg: Development of muscle 
acetylcholinesterase." 

.4 loss and a gain 

On August 13, 1971, Gerard Swope, Jr., announced his retirement as Chairman of the 
Board of Trustees. On that occasion I read a statement prepared jointly by P. B. 
Armstrong, H. B. Steinbach and myself. It read, in part, 

"A man's career is usually reckoned in terms of his achievements; by that reckoning 
Jerry's leadership has been exceptional. For nearly two decades we have benefited 
from his wisdom, two decades marked by material progress, by new buildings, and by 
new programs, all of which have been advanced with a very keen eye tow r ard preserving 
the high ideals of the Laboratory. Possibly it is unfair to single out one achievement 
for special mention, but his leadership at all stages in the development of the dormitory- 
dining hall was truly exceptional. However, the picture of Jerry Swope shines with a 
special luminosity that scintillates not just from these definitive symbols of progress, 
but from his unique combination of personal qualities his warmth and kindness and 
his understanding of the Laboratory and its people. He has understood the lively, 
independent mind and the factors necessary for their interaction. He has worked 
toward the evolution of a community receptive to new ideas. 

Jerry has helped to train, and has guided three directors, all of whom have been 
pleased to serve under his stewardship. No director could have hoped for a more 
productive association. 

Jerry has provided wisdom and practical advice, occasionally with a disconcerting 
ability to isolate weak points for scrutiny in the light of legal logic. We owe very much 
to Jerry Swope and as recipients of his guidance in the past we hope to lay claim to his 
continued wisdom in the future. To that end, however, we must take steps to provide 
the opportunity, for "In a busy life no convenient time ever comes to go visiting in 
cold blood. Some pragmatic stimulant is necessary." Boswell observed of his hero, 
Dr. Johnson, "... on clean-shirt day he went abroad and paid visits." Therefore 
the Trustees have this day elected Jerry S\vope Honorary Chairman of the Board of 
Trustees." 

Many members of the Corporation became acquainted with our ne\v Chairman, 
Denis Robinson, even before his election. His penetrating questions at the Symposium 
on National Policy and the Life Sciences and his lively participation in a wide range of 
Laboratory affairs provided evidence of his understanding of the role and needs of the 



REPORT OF THE DIRECTOR 13 

scientific community in Woods Hole. For those who may not have an immediate 
opportunity to get acquainted with him, here is a "thumbnail sketch." One of the 
nation's most distinguished electrical engineers (Ph.D., University of London), he 
worked in laboratories at the University of London, MIT and the University of Birming- 
ham (England) until 1946 when he founded, and for 24 years served as President of, 
High Voltage Engineering Corporation, where he is now Chairman of the Board. 
Among his many honors, he is a Fellow of the American Academy of Arts and Sciences, 
the American Physical Society and the Institution of Electrical Engineers (London), 
and a member of the National Academy of Engineering. His association with the 
Laboratory is especially timely because of his deep concern about the recycling of 
wastes as they affect our water resources, and with problems arising out of the siting of 
power plants. Denis and his wife Alix are "nearest neighbors" to MBL Beach, their 
summer home being located on Gosnold Road. 

The flow of energy 

In a tribute to J. Walter Wilson, Henry Wriston remarked that Wilson "did not 
look to the president to supply all the energy." Reflecting on his own years of service 
as President of Brown University he observed that the office is, inescapably, an energy 
post, and spoke of the "corporate inertia" of the faculty. In a large and complex 
organization like the Laboratory, not all the moving vigor can flow ceaselessly from the 
Director. It needs to be generated and manifested at every level. Perhaps the most 
disturbing consequence of our failure to attract major institutional support is that 
younger investigators may find it increasingly difficult to come to the Laboratory. 
We must increase the number of promising young scientists in our midst, and open 
channels for their participation in our affairs. We must look to tomorrow's contribu- 
tors, who will keep us abreast of the host of novel challenges that will surely occupy 
the Laboratory in the future. 

I would close with a thought I expressed on another recent occasion. It is especially 
fitting in this context. 

Not long ago I had the privilege of contributing one of two prefaces (the other 
contributor being Peyton Rous) to Paul Weiss's Dynamics of Development: Experiments 
and Inferences. I often think that every critical review and every annual report should 
have authors from two generations. We see what we are conditioned to see. My glimpse 
of the Laboratory tomorrow is only one of many possible visions. 

In Between Pacific Tides, John Steinbeck put it most eloquently: "There is in our 
community an elderly painter of seascapes who knows the sea so well that he no longer 
goes to look at it when he paints. He dislikes intensely the work of a young painter who 
sets his easel on the beach and paints things his elder does not remember having seen." 

1. MEMORIALS 
HAROLD SELLERS COLTON 

Bv WILLIAM F. DlLLER 

Dr. Harold Sellers Colton, Emeritus Professor of Zoology at the University of Pennsyl- 
vania, died at Flagstaff, Arizona on December 29, 1970. He was a native of Philadelphia 
where he was born on August 29, 1881. He received his B.A. degree from the University 
of Pennsylvania in 1904 and his M.A. in 1906. He then became a student of two 
distinguished members of the zoology staff, Dr. J. Percy Moore and Dr. E. G. Conklin. 
Under their guidance he completed a doctoral thesis entitled "Some effects of environ- 
ment on the growth of a pond snail Lymnaea columella Say," published by the Academy 



14 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

of Natural Sciences in Philadelphia. He was awarded the Ph.D. degree by the Uni- 
versity in 1908. Dr. Colton was in the summer course in zoology at the Marine Bio- 
logical Laboratory in 1905, was elected a member of the corporation in 1908, and re- 
tained his interest in MBL into his retirement. 

With the exception of two years (1918-1919) in the Army, where he served in the 
Intelligence Service with the rank of Captain, he continued his association with the 
Department of Zoology at the University of Pennsylvania, and served for a number of 
years as head of the course in elementary zoology. In 1926, as the result of the death 
of a son, he moved to Arizona. Here he threw himself with enthusiasm into studies 
of the geography, geology and anthropology of northern Arizona, as well as into participa- 
tion in many civic projects and organizations. Adjacent to his home he established a 
small laboratory known as the San Francisco Mountain Station of the University of 
Pennsylvania, devoted to the ecology of the local fauna. 

At the University of Pennsylvania he is perhaps best known for his outstanding 
service as director of the course in introductory zoology, where he made many innova- 
tions; and as the author of publications on educational statistics. Those who, like 
myself, were privileged to work under his direction as young instructors in the ele- 
mentary course have remembered him through the years as an outstanding educator 
and as a charming, energetic and stimulating leader whose example has been an inspira- 
tion throughout their own teaching careers. His research interests during his active 
tenure at the University included the ecology of fresh water and marine molluscs and 
excretion in Ascidians. 

Dr. Colton's wife, the former Mary Russell Ferrell, was a noted Philadelphia painter 
(one of the so-called "Philadelphia Ten"). Both became greatly interested in encourag- 
ing Indian arts and crafts and in the prehistoric ceramics of the area. Together with 
her, Dr. Colton founded the Museum of Northern Arizona in Flagstaff, and served as 
its Director from 1928-58, continuing as Director Emeritus until his death. He is 
credited with a major part in the founding of craft shows to encourage Indian artists, 
and with the establishment of two National Monuments, Sunset Crater and the Wapatki 
Ruins north of the Crater. He was one of the first scientists to realize that the volcanic 
area had been occupied by prehistoric Indians prior to massive eruptions in 1064-67. 
His efforts led to the preservation of one of the richest archaeological sites on the 
North American continent. 

Dr. Colton was appointed to the staff of Arizona State College in 1926, where he held 
a professorship until 1953, and an Emeritus Professorship from 1953 until his death. 
He was the recipient of the LL.D. degree from the University of Arizona in 1955, and 
the D.Sc. from the Arizona State College in 1958. He was a member of many important 
societies and was the author of more than 240 articles, monographs and books on widely 
varied subjects. His writings include reports on marine zoology, insect morphology, 
museum history, and on the archaeology, geology and atmospheric conditions of 
Northern Arizona. 

He is survived by his wife, one son, Captain J. Ferrell Colton, two granddaughters, 
and four great-grandchildren. Also surviving are two sisters, Mrs. Robert T. \Yilson 
of Tucson, Arizona and Mrs. Robert P. Esty of Ardmore, Pennsylvania. 

M ANTON COPELAXH 
BY P. SEARS CROWELL 

Manton Copeland was born July 24, 1881 in Taunton, Massachusetts and died at his 
home in Brunswick, Maine, May 22, 1971. He received his degrees from Harvard, 



REPORT OF THE DIRECTOR 15 

the Ph.D. in 1908. He married Ruth Winsor Ripley in 1910. His dedication to 
teaching at Bowdoin College from 1908 until retirement in 1947 and his influence on 
students and through them on biology and medicine is attested in many ways. When 
Dr. Alfred C. Redfield was in charge of Zoology at Harvard, he looked into the question 
of where their graduate students came from and found that Bowdoin had sent more in 
Zoology than any other school, including Harvard College. In 1960 former students 
initiated a memorial scholarship fund with a goal of $25,000. Over the years contribu- 
tions of students and also many Woods Hole friends and others built "Fundy," as it 
was named by "Copey," to the hoped for amount. This was a great satisfaction to 
him and reflects the affection of many individuals. Twelve Copeland scholarship 
awards have already been made. During most of his years at Bowdoin, when he was 
head of the Department of Biology, he taught general zoology as a year course, a semester 
course of general botany, and a course called "Genetics and Evolution" popular with 
non-majors as well as biology students. Besides teaching two courses and their labora- 
tories each semester, he usually had one or more students carrying on a research project. 

The citation of the Bowdoin Alumni Council's Alumni Award for Faculty and Staff, 
given in 1966, reads in part: "A citizen of Brunswick, a summer resident of Woods Hole 
on Buzzards Bay, and a collector incarnate of moths, butterflies, worms, sewing birds, 
duck decoys, flowers, books, friends, students, and children." All were cared for 
meticulously. In the case of the sewing birds he produced a taxonomic key. 

He worked at Woods Hole as an investigator, first at the Fisheries, and from 1915 
to 1945 at MBL. He was elected to the Corporation in 1913. His studies dealt with 
speciation in mammals, physiology of mollusks particularly ciliation and chemorecep- 
tion. Probably his most interesting experiments were those in training Nereis (Ne- 
anthes). He demonstrated conditioned reflexes (in the Pavlovian sense) in these worms. 
He was a member of the Society of Mammalogists, Ecological Society of America, 
American Society of Zoologists, American Ornithologists Union, and a Fellow of The 
American Academy. 

In the Woods Hole community he was perhaps best known for his home, "The Roost," 
on the hill north of the bathing beach and the development with the aid of his sons of 
a delightful labyrinth of paths leading to little special gardens, overlooks, and rock 
pools. His three sons and daughter with their families have spent part of each summer 
at "The Roost" and its annex, "The Coop." One of "Copey's" delights was to conduct 
guests through the paths and constructions, perhaps ending with the view from the 
deck of the "Artemisia," his secluded study at the hill's crest, which was fitted out like 
a ship's cabin, or "The Copecabana," a shelter and picnic area above the rocky shore. 
There a posted sign reads: 

"The real purpose of The Copecabana is not for the sipping of cocktails, or the 
cracking of lobster shells. Rather it is to give you some protection from the ele- 
ments and a feeling of seclusion that you may better appreciate and gain more 
happiness from the everchanging picture that lies before you. Your are in one 
of the most beautiful and exciting spots on earth where the land meets the sea. 
Here life is probably more abundant than in any other locality; in fact, it may 
have arisen here. The littoral is never the same. It varies with every tide, 
wave, and wind. . . . Come here alone, or with an understanding companion, 
in the early morning, or when the sun is setting over Penzance. Look before 
you, in front of you, and above and try to understand what it all means. Come 
again and again because what you see is not all that you can see. Eventually 
you may reach the only possible conclusion. In any case you will dream better 
dreams, which is a key to happiness. And that is the real purpose of The 
Copecabana." 



16 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

CHARLOTTE HAYWOOD 
BY CURTIS J. SMITH 

It is never possible to capture the essence of a complex personality in a few words. 
If it were possible, the words used of Charlotte Haywood would sound like the exhorta- 
tions of a slightly old-fashioned schoolmaster: "strength of character," "self-discipline," 
"absolute integrity," and "selfless devotion." No one would deny that Charlotte 
exemplified these virtues, but no one who knew her would allow that such phrases do 
justice to her liveliness and enthusiasm, her love of beauty, or the warmth of her 
friendship. 

She was, in many respects, a fairly typical product of the great patrician tradition 
of New England. As a girl she often accompanied her physician father on his rounds 
in a horse and buggy. She must have absorbed the uncompromising standards of her 
Yankee heritage, along with the appreciation of art and the love of science which marked 
her whole life. 

As an undergraduate at Mount Holyoke College she began an association with Abby 
Turner, who, along with Ann Morgan, carried on the traditions of excellence in science 
under the impetus of the legendary Cornelia Clapp. The association was to continue 
for the rest of Miss Turner's life; and it is one of the tragedies of Charlotte's own life 
that she was never able to find a protegee to continue this worthy succession. 

Those undergraduate days were not all plain living and high thinking! Charlotte 
was always fun-loving and full of the zest for living which remained one of her most 
charming characteristics to the very end. Only a few years before her retirement she 
managed to hitch a ride on Otto Kohler's antique fire engine while it was transporting 
male members of the faculty on a careening tour of the campus during their annual 
post-graduation party. She later remarked, with considerable satisfaction, "It was 
something I've always wanted to do!' 

Endearing as her personal traits were, it is in her career that one finds her greatest 
monument. Between earning her master's degree at Brown University and her Ph.D. 
at the University of Pennsylvania, she returned to Mount Holyoke as an instructor of 
physiology from 1921 to 1924. After receiving her doctorate she taught for three years 
at Vassar, but in 1930 she came back to Mount Holyoke for the career of teaching and 
research that was to continue for the rest of her life. 

In that long span of time a great many of her students have achieved distinction in 
careers of their own. They provide visible testimony to the excellence of her teaching 
and the inspiration of her example. But even more than this, it was in her elementary 
courses, taught not specifically for science students, but as a part of a liberal education, 
that she made her greatest mark. Hundreds of students who got their first, and some- 
times their only, taste of science at her hands, remember her with affection and respect ; 
and few of them could have realized how meticulously the smooth-running laboratories 
were rehearsed with the teaching staff, or how many hours she spent preparing each 
lecture, no matter how familiar she was with the material. Even when the enrollment 
exceeded two hundred, she would know the name of every student in her class, and no 
returning alumna was unrecognized. There are many roads to immortality; and in 
the many hundreds of students that knew Charlotte Haywood her memory lives on. 

Although her love for teaching took first place in her life, Charlotte was an active 
and productive research scientist as well. Many summers were spent at her beloved 
Woods Hole, at the marine biological station, carrying on her studies in respiratory 
physiology. Dr. Helen W. Kaan, her classmate and close friend, recalls that beginning 
with the summer of 1920, Dr. Haywood spent virtually every summer (except those spent 
in European laboratories) in research and teaching at the Marine Biological Laboratory 
.in Woods Hole. After her retirement in 1961 she continued faithfully to attend the 



REPORT OF THE DIRECTOR 17 

Annual Corporation Meetings at the Marine Biological Laboratory, and was present 
at the August, 1970, meetings. She was one of the first woman members of the Ameri- 
can Physiological Society, and was known and respected by a great many of the scientific 
"establishment." After her retirement she continued her work on her scientific publica- 
tions, and attended seminars whenever she found time in her busy life. 

Although her whole professional life had been characterized by an extraordinary 
devotion to her students and her work, upon her retirement Charlotte revealed new 
talents and found new outlets for her restless mind. Always an ardent traveler, she 
was finally able to extend her wandering to a trip around the world. Her life-long 
love of flowers was expressed in a new-found skill in photography. She continued her 
regular attendance at concerts, plays, and lectures; and she found more time for her 
friends. 

Throughout her life her contacts with students, friends, and associates were charac- 
terized by an ingrained and spontaneous graciousness. She was uncompromising in 
her personal standards, but forgiving of others. Perhaps she typified her era, but her 
gentle nobility of spirit is a quality needed by every era. Her passing sadly diminishes 
our world. 

FAITH STONE MILLER 
BY S. MERYL ROSE 

Faith Stone was born in Newton, Massachusetts on September the twenty-third, 1908 
to Alaric Maxwell Stone and Ruth Taylor Stone. Hers was a full life as daughter, wife, 
mother, friend, research worker and teacher until her sudden death on June 11, 1971, 
in Falmouth, Massachusetts. 

She grew up in and attended school in Newton. After graduation from Newton 
High School she entered Mount Holyoke College with a College Entrance Board Com- 
petitive Scholarship for New England. From her sophomore year through till gradua- 
tion in 1930 she held an Alumnae Association Scholarship. During the next two years 
she served as an Assistant in Zoology at Connecticut College for Women. After that 
she went on to the University of Chicago where she received the Ph.D. in Zoology in 
1937. It was at Chicago that the great collaboration between Faith Stone Miller and 
her husband, James A. Miller, Jr., began. They were both students of Charles Manning 
Child and began their studies on regeneration under him. One of the many things 
they observed was that decreased temperatures prolong life. Their research evolved 
into their well known studies demonstrating that hypothermia can cause recovery 
from asphyxia in the newborn. 

There were some important years when most of her time was spent with her children, 
David and Janet. When they became self-sufficient she returned to academic work in 
1948 as a part-time Research Assistant working with her husband at Emory University. 
There she also became the Director of Basic Sciences for Nurses. Then in 1960 came 
the move to Tulane University when her husband became the Chairman of the Depart- 
ment of Anatomy. She advanced from Assistant Professor to Associate Professor of 
Anatomy and became the recognized and respected unofficial Assistant Chairman. 
Together Dr. Faith Miller and her husband built an excellent department including an 
outstanding center for the training of anatomists. It was in her work with graduate 
students that Dr. Miller was at her best as a teacher. With quiet, kindly humor and 
sincere interest she brought out the best in them. She always had as much time as 
required for their academic and personal problems. 

There were awards and prizes along the way. One of the early ones was the Collecting 
Net Award for Excellence in Invertebrate Zoology at the Marine Biological Laboratory. 
The laboratory, where she became a member of the Corporation, their home in Woods 
Hole and its environs grew in importance to her. She shared with her husband in 1959 



18 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

the Southeastern Biologists Research Prize and the Sigma Xi Research Citation at 
Emory University. She was the Martha Catching Enochs Fellow of the American 
Association of University Women during 1957-1958 in London and in 1962 was a 
Fulbright Research Scholar in Finland. These years, including a period in Sweden, 
were spent with her husband on the valuable studies which established that infants 
asphyxiated during birth could be revived without subsequent ill effects. The results of 
the Millers' research were reported in many papers and at national and international 
meetings. Confirmation of the beneficial effects of hypothermia in overcoming asphyxia 
has come from investigators in several European countries. 

Dr. Faith Miller was a member of the Society of Sigma Xi, the American Association 
of Anatomists, the American Society of Zoologists, the Association of Southeastern 
Biologists and the Southern Society of Anatomists. 

Visits to foreign lands were opportunities for collecting beautiful old objects. Many 
of these are in their tastefully restored home in old New Orleans where she served as 
a gracious hostess. Dr. Faith Miller's unique blend of knowing kindness, quiet humor 
and personal interest in her associates has enriched the lives of many of us. 

EMIL WITSCHI 
BY JOYCE BRUNER-LORAND 

The life motif of Emil Witschi was his love of nature and his untiring and selfless 
devotion to scientific research. Professor Witschi was born in a small village near Bern, 
Switzerland, February 18, 1890. He spoke fondly of his early years spent in the pastoral 
surroundings of alpine valleys which offered him the luxury of contemplation of the 
many forms of developing life. By the time he went to Munich to pursue his doctoral 
studies under the guidance of Richard Hertwig, he was thoroughly indoctrinated as a 
naturalist. He received his doctorate in 1913, and published the work with which he 
was most intimately identified, the inductor theory of sex differentiation in 1914. From 
his studies on sex differentiation in Rana temporaries, he very early concluded that all 
embryos and the primordial germ cells are bipotential, that they may develop in either 
the male or female direction, that the alternative of male or female differentiation 
depended upon both genetic and non-genetic factors. His demonstration that the 
embryonic gonadal cortex acts as inductor of female differentiation and that the medulla 
induces male differentiation is basic to all considerations of the problem of human 
intersexuality. 

Professor Witschi left Switzerland in 1926 and came to America as a Rockefeller 
Foundation Fellow. He had barely set foot in the United States when he was requested 
to deliver an evening lecture at the Marine Biological Laboratory, an event which he 
often recalled with a mixture of terror speaking in a new language, and with pleasure- 
as it marked the beginning of association with the MBL which was to last almost half a 
century. 

In 1927 Professor Witschi joined the faculty of the State University of Iowa. He was 
particularly fond of its beautiful setting amid the rolling hills of the Iowa countryside 
overlooking the Iowa river. It reminded him of his native Bernese Oberland. His 
devoted wife Martha and his two children joined him there and the decision was made 
to assume American citizenship. Few universities have been so privileged to count 
among its faculty a man of such outstanding and diversified achievement. He was a 
dedicated teacher. Those who attended Professor Witschi's embryology lectures can 
hardly forget his ability as an artist. The most intricate three dimensional drawings 
were swiftly and deftly executed. His lectures were truly a unique intellectual and 
artistic experience. His great knowledge in the field of embryology found expression 
in a textbook "The Development of Vertebrates," published in 1955. 



REPORT OK THE DIRECTOR 1 <) 

In the laboratory. Professor Witschi's work covered a broad range of topics, pursued 
with endless enthusiasm, devotion and the highest degree of excellence. He understood 
the importance of comparative biology, and his work in endocrinology and sex differ- 
entiation was carried out on more than 30 forms, ranging from invertebrates to man. 
His contributions have been fundamental to the understanding of many fields of en- 
deavor: human sex development genetic, developmental and hormonal aspects of 
gonadal dysgenesis and sex inversion in man; the migration of germ cells in the human 
embryo; the comparative aspects of pituitary gonadotrophins ; the evolution of endo- 
crine reactions hormonal control of feather and bill coloration in birds; teratogenesis 
and fetal abnormalities overripeness and temperature effects in the amphibian egg; 
genetics the mechanism of sex inheritance in amphibians; behavior studies on the 
herring gull ; underwater hearing the basilar papilla of the amphibian ear. 

Emil Witschi was the recipient of many honors. We can mention the Fred Conrad 
Koch Award and medal, the honor of highest distinction conferred by the Endocrine 
Society, and the degree of honorary Doctor of Medicine conferred on him by the 
University of Basel on the occasion of the 500th anniversary of its founding. He was 
doubtlessly pleased by these and the many other honors which came his way. But for 
Emil Witschi satisfaction lay in the inspiration he provided to his students, over 45 men 
and women who earned their Ph.D. degree with him, and to the many other researchers 
who by virtue of his example and encouragement would continue with the same vitality 
and dedication for which we shall always remember him. 

For those of us who were privileged to enjoy the warm hospitality of his home so ele- 
gantly presided over by his gracious wife Martha, the memory will always be cherished. 

EDGAR /WILLING 
Hy MAC V. EDDS, JR. 

Edgar Zwilling was born in Pittsburgh on February 1, 1913 ; he died on July 23, 1971. 
His parents were Russian expatriates; his father, a blacksmith, lived only until Edgar 
was five. The family moved then to Brooklyn where Edgar grew up. He saw the 
prosperity of the 20's only from a distance; his boyhood was one of harshness, struggle, 
and hard-won reward. 

Edgar attended Brooklyn College from which he was graduated in 1933; there he 
had developed an interest in biology, and he turned to graduate study in embryology 
at Columbia University where under the guidance of Lester Barth he received the 
Ph.D. in 1940. For the balance of his life, Edgar served three institutions with devotion 
and steadfastness: The University of Connecticut as an investigator in the Animal 
Genetics Department of the Storrs Agricultural Experiment Station; Brandeis Uni- 
versity as professor and chairman of Biology; and the Marine Biological Laboratory 
as student, instructor, investigator, committee member, and tireless advocate. More 
briefly, Edgar also served the National Science Foundation as Program Director of 
Developmental Biology, the National Institutes of Health as a Training Grant Panel 
member, the Society for Developmental Biology as its president, the American Society 
of Zoologists as chairman of the Developmental Biology Division. One of his last 
honors came in election to the American Academy of Arts and Sciences. 

As these marks of recognition attest, Edgar came to be held in high regard for his 
contributions to developmental biology. His forte was experimental morphogenesis 
practiced at tissue and organ levels. But he was no stranger to the cellular and molecu- 
lar approaches that evolved during his academic life, and he used them skillfully to 
contribute understanding to the genesis of form at higher levels. Considering his nearly 
single-minded refusal to be distracted by merely fashionable lines of research, Edgar 
worked on a surprising diversity of developmental problems, including more or less in 



20 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

chronological order, the origin of the nose and the ear in the frog embryo, regeneration 
in hydroid coelenterates, etiology of developmental anomalies, and his major work 
cell and tissue interactions in the development of the chick limb. In now classical 
papers starting in the middle 1950's, Edgar analyzed the outgrowth of limb bud mesoderm 
under the influence of the apical ectodermal ridge, as well as the reciprocal role of the 
mesoderm in maintaining and determining the form of the ridge. These studies, note- 
worthy for their ingenuity and for the technical skill on which they depended, led Edgar 
to investigations of the emergence and fixation in the limb bud of the morphogenetic 
quality of "limbness," that is, the capacity to form a limb. He showed that this prop- 
erty is already present even before a definite limb bud forms; that it is a result of pat- 
terned cell-cell interactions; that it survives the disaggregation of limb mesoderm cells 
to reappear after their reaggregation ; and that it persists until individual cells begin 
their overt differentiation into muscle, cartilage, and the like. The transition from a 
morphogenetic to a cytodifferentiative phase occurs gradually over several hours; it is 
preceded by the emergence in very small amounts of those end product molecules and 
enzyme activities that will subsequently characterize the full blown cytodifferentiative 
phase. 

Since these contributions as well as their significance have already been reviewed in 
earlier biographical essays (Edds, 1972, Developmental Biology, 28: 1; Saunders, 1971, 
Developmental Biology, 26: 165), they will not be examined further here except to recall 
Edgar's preoccupation with the double thesis that, first, the emergence of form, 
especially at the organ level, is the central mystery of development, and its unravelling 
is the main goal of developmental biology; and, second, that obsession with any single 
analytical approach will always result in falling short of the goal. As he put it, "a 
complete understanding of the ontogeny of functional form will eventually relate the 
molecular, cellular, or supracellular phenomena responsible for the elaboration of form 
to the synthetic activities of the fully differentiated cells." In this uncompromising 
commitment to that central theme, he left a memorable heritage of basic contributions 
to embryology. In the warmth and generosity of his equal commitment to family, to 
friends, to students, to colleagues, and to the institutions he served, Edgar Zwilling left 
a large, indelible mark on hundreds of lives. 



2. THE STAFF 
KMBRYOLOCA' 

I. CONSULTANTS 

EVERETT ANDERSON, Professor of Biology, University of Massachusetts 

ANTHONY CLEMENT, Professor of Biology, Emory University 

DONALD P. COSTELLO, Professor of Zoology, University of North Carolina 

II. INSTRUCTORS 

MALCOLM S. STEINBERG, Professor of Biology, Princeton University, in charge of course 

MAX BURGER, Associate Professor of Biology, Princeton University 

RALPH HINEGARDNER, Associate Professor of Biology, University of California, Santa 

Cruz 

HANS LAUFER, Associate Professor of Zoology, University of Connecticut 
ERIC DAVIDSON, Associate Professor of Biology, California Institute of Technology 
GARY FREEMAN, Assistant Professor of Biology, University of California, San Dieg 



REPORT OF THE DIRECTOR 



21 



III. SPECIAL LECTURERS 

JOHN ARNOLD, Associate Professor of Cytology, University of Hawaii 
RAYMOND RAPPAPORT, Professor of Biology, Union College 
PAUL B. WEISZ, Professor of Biology, Brown University 

IV. LABORATORY ASSISTANTS 

ANTHONY W. SHERMOEN, Wesleyan University 
DAVID M. MIYAMOTO, Duke University 



M. S. STEINBERG 
J. P. TRINKAUS 

R. HlNEGARDNER 

M. S. STEINBERG 
E. ANDERSON 
RAYMOND RAPPAPORT 

H. LAUFER 

ROBERT BRIGGS 
RONALD H. REEDER 

YOSHIAKI SUZUKI 
ERIC DAVIDSON 
RALPH HINEGARDNER 
LAJOS PIKO 
MAX BURGER 
ANTHONY CLEMENT 

JOHN ARNOLD 
GARY FREEMAN 

BETH BURNSIDE 
M. S. STEINBERG 
HERBERT M. PHILLIPS 



ANTONE JACOBSON 
JOHANNES HOLTFRETER 
PAUL B. WEISZ 
DAVID S. BARKLEY 

VIKTOR HAMBURGER 



V. LECTURES 

Introduction to the course 

Development of coelenterates 

Control processes in coelenterate development 

Analysis of teleost development (I) 

Analysis of teleost development (II) 

Echinoderm development: egg to pluteus 

Echinoderms: life cycle and experimental embryology 

Morphogenetic phenomena in sponges 

Ultrastructure of oocytes 

Cytokinesis I 

Cytokinesis II 

Hormonal control of differentiation 

Chromosomal puffing: its developmental significance 

Genetic control of the egg cytoplasm 

Isolation and transcription of the genes for ribosomal 
RNAs 

Induction and sequencing of the mRXA for silk tibroin 

Sequence homology studies with eukaryotic nucleic acids 

Evolution of cellular DXA content 

Fertilization 

Cell surface changes in neoplasia and during cytokinesis 

Early development of spiralians 

Experimental analysis of spiralian development 

Experimental studies on cephalopod development 

Organization and early development of the ascidian egg 

The cellular basis of asexual reproduction in ascidians 

The roles of microtubules and microtilaments in morpho- 
genesis 

Does differential cell adhesion govern self-assembly proc- 
esses in morphogenesis? I. Behavioral evidence 

II. Precise formulation and direct physical testing of the 
differential adhesion hypothesis 

III. Application of the differential adhesion hypothesis to 
early amphibian morphogenesis 

Experiments on the control of organ determination 

Embryonic induction and morphogenetic fields 

The significance of larvae 

Pattern formation in aggregates of developing neural 

tissue 
Neurogenesis and the origins of behavior 



22 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

VI. POST COURSE PERIOD 

THOMAS ROTH Physiological and structural evidence for protein transport 

into oocytes 
ROBERT O. BECKER Stimulation of partial limb regeneration in mammals 



PHYSIOLOGY 

I. CONSULTANTS 

ALBERT SZENT-GYORGYI, Director, The Institute of Muscle Research, Marine Biological 

Laboratory 

\Y. D. McELROY, National Science Foundation 
|. WOODLAND HASTINC.S, Professor of Biology, Harvard University 

II. INSTRUCTORS 

ANDREW G. SZENT-GYORGYI, Professor of Biology, Brandeis LIniversity, in charge of 

course 

RODERICK K. CLAYTON, Professor of Biophysics, Cornell University 
LAWRENCE GROSSMAN, Professor of Biochemistry, Brandeis University 
HUGH E. HUXLEY, Medical Research Council, Laboratory of Molecular Biology, 

Cambridge, England 

PETER C. NEWELL, Department of Biochemistry, University of Oxford, Oxford, England 
DAVID A. YPHANTIS, Professor of Biology, University of Connecticut 

III. SPECIAL LECTURERS 

HARLYN O. HALVORSON, Professor of Bacteriology, L T niversity of Wisconsin 
SHINYA INOUE, Professor of Biology, University of Pennsylvania 

IV. STAFF ASSOCIATES 

RAYMOND E. STEPHENS, Department of Biology, Brandeis University 

ANNEMARIE WEBER, Department of Biochemistry, St. Louis University 

RICHARD J. PODOLSKY, Laboratory of Physical Biology, Xational Institute of Arthritis 

and Metabolic Diseases 

JACOB FRANKE, Department of Biology, Brandeis University 
MICHAEL JOHNSON, Department of Biophysics, University of Connecticut 
WALTER F. STAFFORD, III, Department of Biophysics, University of Connecticut 
ROBERT HASELKORN, Department of Biophysics, University of Chicago 
WILLIAM LEHMAN, Department of Biology, Brandeis University 
ANDREW BRAUN, Department of Biochemistry, Brandeis University 

V. RESEARCH ASSISTANTS 

RUTH HOFFMAN, Department of Biology, Brandeis University 

EVELYN W T OOD, Department of Biophysics, Cornell University 

PAUL SAPIN, Boston University 

GAIL CLINTON, Department of Biology, University of California at La Jolla 

JOAN NEWELL, Department of Biochemistry, University of Oxford, Oxford, England 

VI. COURSE ASSISTANT 
MARY VAN HOLDE, University of Oregon 



REPORT OF THE DIRECTOR 



23 



ANDREW G. SZENT-GYORGYI 
ANNEMARIE WEBER 
ANDREW G. SZENT-GYORGYI 
DAVID A. YPHANTIS 
JOHN G. NICHOLS 

DAVID A. YPHANTIS 
LAWRENCE GROSSMAN 
JEFFRIES WYMAN 

LAWRENCE GROSSMAN 
RODERICK K. CLAYTON 

RICHARD J. PODOLSKY 
HENRY R. MAHLER 
EDWARD ADELBERG 
MAURICE S. Fox 
PETER C. NEWELL 

HUGH E. HUXLEY 
SHINYA INOUE 
R. E. STEPHENS 

ANTHONY C. H. DURHAM 
MICHAEL F. MOODY 
ROBERT HASELKORN 
HARLYN O. HALVORSON 
LEWIS G. TILNEY 
LASZLO LORAND 
DAVID SHEMIN 
ALBERT SZENT-GYORGYI 
SEYMOUR S. COHEN 

AKIRA KAJI 
CYRUS LEVINTHAL 
BERNARD D. DAVIS 

jEAN-PlERRE CHANGEUX 

JONATHAN B. WITTENBERG 
SALVADOR E. LURIA 
GUSTAV V. R. BORN 
LAWRENCE B. COHEN 
J. WOODLAND HASTINGS 

ADOLPH I. COHEN- 
RUTH HUBBARD 
WILLIAM HAGINS 
ED KRAVITZ 



VII. LECTURES 

Aspects of the chemistry of contraction 

Regulation of contraction 

The proteins, and their assembly in molluscan muscles 

Physical biochemistry I 

Specific connection and regeneration patterns of sensory 
and motor nerve cells of the leech nervous system 

Physical biochemistry II 

Physical biochemistry III 

Mechanism of replication in vivo and in vitro I 

Mechanism of replication /;/ vivo and in vitro II 

Principles of linkage 

Illustrations in biological macromolecules 

Enzymatic repair of DNA 

Photosynthesis I 

Photosynthesis II 

Control of contraction 

Mitochondrial DNA and mitochondria! mutation 

Transfer replication of DNA in E. coli 

On integration of DNA in bacterial transformation 

Developmental control in slime molds I 

Developmental control in slime molds II 

Structural aspects of muscle contraction I 

Structural aspects of muscle contraction II 

Biophysical analysis of mitosis in living cells I 

Biophysical analysis of mitosis in living cells II 

Biochemistry of microtubular systems I 

Biochemistry of microtubular systems II 

How tobacco mosaic virus assembles 

Structure and contraction of the bacterial phage tail 

The physiology of the bacteriophage T 4 development 

Meiosis in yeast 

Microtubules in development of cell forms 

Enzyme catalyzed protein assemblies, fibrin 

Enzymes in heme synthesis 

Water, motion and muscle 

The physiology, biochemistry and molecular biology of 
polyamines 

On the mechanism of protein synthesis 

Symmetry in brains of little animals 

The ribosome-polysome cycle 

Studies of cholinergic receptor protein in the electric eel 

What does myoglobin do? 

Colicins 

Adhesion of thrombocytes and leucocytes 

Optical studies of action potentials 

Bioluminescence : endosymbiosis in the ponyfish and sub- 
unit functions in luciferase 

Rods and cones 

Photochemistry of visual pigments 

Studies on exitation in outer segments of rods 

Studies on synaptic chemistry in single nerve cells 



24 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

DAVID HUBEL Architecture and integration in the visual cortex of the 

brain 
NIGEL W. Dow Neurophysiology of color vision 

VIII. SPECIAL LECTURE 
ALLAN WEED Comparative studies of the light chains of myosin 

EXPERIMENTAL MARINE BOTANY 

I. CONSULTANTS 

STERLING B. HENDRICKS, .National Academy of Sciences 
BESSEL KOK. Research Institute for Advanced Studies 
LAWRENCE GOGORAD, Harvard University 

II. INSTRUCTORS 

HAROLD W. SIEGELMAN, Brookhaven National Laboratory, in charge of course 

ROBERT L. GUILLARD, Woods Hole Oceanographic Institution 

SYNNOVE LIAAEN JENSEN, Norwegian Institute of Technology 

ARNE JENSEN, Norwegian Institute of Technology 

FRANK A. LOEWUS, State University of New York at Buffalo 

ANTHONY G. SAX PIETRO, Indiana University 

JEROME A. SCHIFF, Brandeis University 

MICHAEL J. WYNNE, University of Texas 

III. SPECIAL LECTURERS 

JOSEPH MASCARENHAS, State University of New York at Albany 

N. KEITH BOARDMAN, C.S.I.R.O., Australia 

R. P. LEVINE, Harvard University 

SHIMON KLEIN, Hebrew University, Israel 

DONALD J. PLOCKE, Boston College 

MORDHAY AVRON, Weizmann Institute, Israel 

OTTO KANDLER, University of Munich, Germany 

W. W. YOUNGBLOOD, Woods Hole Oceanographic Institution and Florida Technological 

University 

BJORN LARSON, Norwegian Institute for Seaweed Research, Norway 
NORMAN KRINSKY, Tufts University School of Medicine 
ARTHUR STERN, University of Massachusetts, Amherst 

IV. RESEARCH ASSOCIATES 

MARTIN GIBBS, Brandeis University 
CARL A. PRICE, Rutgers University 

V. RESEARCH ASSISTANTS 

GEORGE WAGNER, State University of New York at Buffalo 
RICHARD WETHERBEE, University of Michigan 

VI. LECTURES 

H. W. SIEGELMAN Photobiologically active plant chromoproteins 

ARNE JENSEN Chemistry of the algal classes 

SYNNOVE LIAAEN-JENSEN Carotenoids-general aspects 

Comparative biochemistry of marine carotenoids 



REPORT OF THE DIRECTOR 



25 



ARNE JENSEN 
ROBERT L. GUILLARD 



MICHAEL J. WYXXE 
JEROME A. SCHIFF 
MICHAEL J. WYNNE 

JOSEPH MASCARENHAS 
MICHAEL J. WYNNE 

N. KEITH BOARDMAN 
JEROME A. SCHIFF 

R. P. LEVINE 
JEROME A. SCHIFF 
SHIMON KLEIN 

ANTHONY SAN PIETRO 



DONALD J. PLOCKE 
MORDHAY AVRON 

OTTO KANDLER 
MARTIN GIBBS 
W. W. YOUNGBLOOD 
F. LOEWUS 
CARL A. PRICE 
F. LOEWUS 
BJORN LARSEN 
NORMAN KRINSKY 
ARTHUR STERN 



Perspectives in algal chemistry 

Utilization of seaweeds 

Cultures as tools for the study of phytoplankton ecology 

Physiological races of marine planktonic diatoms 

Limitation of phytoplankton growth by low nutrient levels 

Introduction to the benthic marine algae: chlorophyta 

Sulfate metabolism in algae 

Introduction to the benthic marine algae: rhodophyta and 

phaeophyta 

Intracellular structures and their movements in plant cells 
Vertical zonation and distribution patterns along the 

atlantic coast 

The photosynthetic process 
Plastid structure and evolution 
Plastid development and inheritance 
Genetics of photosynthesis and the chloroplast 
Evolution of photosynthetic pigment systems 
Comparative aspects of chloroplast development in 

Euglena and higher plants 
Photosynthetic electron transport and photophosphoryla- 

tion. I. Introduction 

II. Components of the system 

III. Current view 

Altered ribosomes in zinc- and iron-deficient micro- 
organisms 

Electron transport and carbon metabolism in photo- 
synthesis 

Physiology and biosynthesis of Hamamelose 

Photorespiration 

Hydrocarbons in algae 

Carbohydrate interconversions involving inositol 

Zonal centrifugation and particle separation 

A functional role for carbohydrate exudate of plants 

Biosynthesis of alginic acid 

Protective function of carotenoid pigments 

Photophosphorylation and ATPase activity in mature and 
developing chloroplasts 



EXPERIMENTAL INVERTEBRATE ZOOLOGY 

I. CONSULTANTS 

FRANK A. BROWN, JR., Professor of Zoology, Northwestern University 

C. LADD PROSSER, Professor of Physiology, University of Illinois 

CLARK P. READ, Professor of Biology, Rice University 

ALFRED C. REDFIELD, Woods Hole Oceanographic Institution 

W. D. RUSSELL-HUNTER, Professor of Zoology, Syracuse University 

I 1. INSTRUCTORS 

JAMES F. CASE, Professor of Biology, University of California, Santa Barbara, in charge 

of course 

GARTH CHAPMAN, University College, London 
ALAN GELPERIN, Assistant Professor of Biology, Princeton University 



26 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



DAVID C. GRANT, Assistant Professor of Biology, Davidson College 

MICHAEL J. GREENBERG, Associate Professor, Florida State University 

JOSEPH B. JENNINGS, Department of Zoology, University of Leeds 

CHARLOTTE P. MANGUM, Associate Professor of Biology, College of William and Mary 

JAMES G. MORIN, Assistant Professor of Zoology, University of California, Los Angeles 

DOROTHY M. SKINNER, Biology Division, Oak Ridge National Laboratory 

III. SPECIAL LECTURERS 

THOMAS J. M. SCHOPF, University of Chicago 

G. REYNOLDS, Princeton University 

WILLIAM STEWART, University of California, Santa Barbara 

R. L. PARDY, University of California, Los Angeles 

DEMOREST DAVENPORT, VJniversity of California, Santa Barbara 

JOHN H. TODD, Woods Hole Oceanographic Institution 

KENNETH D. ROEDER, Tufts University 

FOTIS KAFATOS, Harvard University 

AUDREY E. V. HASCHEMEYER, Hunter College 

JEFFREY M. CAMHI, Cornell University 

IV. ASSISTANTS 

ARNOLD G. EVERSOLE, Syracuse University 
EVE C. HABERFIELD, University of Rhode Island 
GEORGE A. KAHLER, III, Rice University 



J. CASE 
J. MORIN 

G. CHAPMAN 
T. SCHOPF 

C. MANGUM 
M. GREENBERG 

D. SKINNER 

G. CHAPMAN 

J. CASE 

DEMOREST DAVENPORT 

JOHN H. TODD 
C. MANGUM 

M. GREENBERG 



C. MANGUM 
A. GELPERIN 



Y. LECTURES 

Introduction 

Invertebrate phylogeny 

Porifera and Cnidaria 

Cnidaria and ctenophores 

Flatworms, nemerteans, aschelminthes 

Ectoprocts 

Annelida 

Mollusca I 

Mollusca II 

Arthropoda I 

Arthropoda II 

Echinoderms 

Protochordates 

Computerization studies of the orientation of microscopic 

organisms 

An ethology of stress environments 
Respiration. I. Exchange 
Respiration. II. Transport 

Patterns of circulation among the invertebrates 
Hearts and visceral muscle: the way to a clam's heart is 

through its rectum 

Some aspects of comparative muscle physiology 
Temperature adaptation 
Regulation of feeding 



REPORT OF THE DIRECTOR 



27 



J. JENNINGS 



FOTIS KAFATOS 
J. MORIN 
A. GELPERIN 



J. CASE 

KENNETH D. ROEDER 

J. CASE 

G. REYNOLDS 

J. MORIN 

WILLIAM STEWART 

D. SKINNER 

J. CASE 
AUDREY E. V. 

HASCHEMEYER 
JEFFREY M. CAMHI 
R. L. PARDY 



Alimentary systems 

Digestive physiology with particular reference to acoe- 

lomates I. 
Digestive physiology with particular reference to acoe- 

lomates II. 

Hormone-initiated cellular differentiation in insects 
Primitive nervous systems 
Complex behavior in simple neural systems 
Endogenous activity and rhythmic behaviors 
Command and executive neurons 
Invertebrate photoreception 
Palps and pilifers 
Invertebrate chemoreceptors 
Methods in the investigation of bioluminescence 
Bioluminescence in the lower metazoa 
Factors influencing larval settlement 
Growth and molting I 
Growth and molting II 
Bioluminescence in higher metazoa 
Studies on the mechanism of temperature acclimation of 

marine organisms 

Diverse functions of the locust flight motor 
Algal symbiosis in coelenterates 



MARINE ECOLOGY 

I. CONSULTANTS 

MELBOURNE R. CARRIKER, Marine Biological Laboratory 
HOWARD L. SANDERS, Woods Hole Oceanographic Institution 
LAWRENCE B. SLOBODKIN, State University of New York at Stony Brook 
ROGER Y. STANIER, University of California at Berkeley 

I 1. INSTRUCTORS 

HOLGER W. JANNASCH, Senior Scientist, Woods Hole Oceanographic Institution, in 

charge of course 

RICHARD W. CASTENHOL/, Professor of Botany, University of Oregon at Eugene 
RALPH MITCHELL, Professor of Applied Microbiology, Harvard University 
SUMNER RICHMAN, Professor of Biology, Lawrence University 
DONALD C. RHOADS, Associate Professor of Geology, Vale University 
EDWARD O. WILSON, Professor of Zoology, Harvard University 

III. SPECIAL LECTURERS 

JELLE ATEMA, Woods Hole Oceanographic Institution 

ERCOLE CANALE-PAROLA, University of Massachusetts at Amherst 

FREDERICK J. GRASSLE, Woods Hole Oceanographic Institution 

J. WOODLAND HASTINGS, Harvard University 

GALEN E. JONES, University of New Hampshire 

EDWARD R. LEADBETTER, Amherst College 

KENNETH NEALSON, Harvard University 

JOHN D. PALMER, New York University 



28 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



SYDNEY C. RITTENBERG, University of California at Los Angeles 

HOWARD L. SANDERS, Woods Hole Oceanographic Institution 

LAWRENCE B. SLOBODKIN, State University of Xew York at Stony Brook 

JOHN M. TEAL, Woods Hole Oceanographic Institution 

JOHN TODD, Woods Hole Oceanographic Institution 

WOLF VISHNIAC, University of Rochester 

RALPH S. WOLFE, University of Illinois at Urbana 

IV. LABORATORY ASSISTANTS 

HERMAN F. BOSCH, Johns Hopkins University 
DENIS CUNNINGHAM, Yale University 



H. W. JANNASCH 



E. R. LEADBETTER 
S. C. RITTENBERG 
R. S. WOLFE 
R. MITCHELL 



J. W. HASTINGS 

K. NEALSON 

R. W. CASTENHOLZ 



E. CANALE PAROLA 
S. RICHMAN 



J. M. TEAL 
H. F. BOSCH 



V. LECTURES 

Introduction to microbial ecology 

Marine microbiology I and II 

Continuous culture in microbial ecology 

Theory and practice of continuous culture techniques 

Microbiology of the Black Sea 

Ecology of photosynthetic bacteria 

How organisms make a living 

Methods of microbial ecology 

Microbiology of marine sediments 

The microbial sulfur cycle 

Anaerobic transformations in the cycle of matter 

Anaerobic microorganisms in marine sediments 

Intermicrobial predation 

Ecological aspects of bacterial chemotaxis 

Attachment of bacteria to surfaces 

Unusual iron and manganese bacteria 

Marine fouling 

Bacterial aggregation 

Microbial approaches to water pollution control 

Symbiosis of luminescent bacteria 

Problems in the classification of marine bacteria 

Photosynthesis and productivity in the sea 

Light in natural waters; effects on photosynthesis and 
growth 

Inorganic nutrients as factors limiting the growth of 
phytoplankton 

Organic factors limiting growth of phytoplankton 

Intertidal zonation of macro-algae 

The ecology of hot springs I and II 

Biology of Spirochaetes 

Intermediary metabolism of Spirochaetes 

Food chains and ecological efficiencies 

Energetics of single species 

Quantitative methods for measuring the feeding of plank- 
tonic Crustacea 

Assimilation of food by zooplankton 

Studies on the feeding behavior of copepods I and II 

Salt marsh ecology 

Experimental work on the Sippewissett salt marsh 

The marine Cladocera 



REPORT OF THE DIRECTOR 29 

D. C. RHOADS Structure and dynamics of henthic invertebrate assem- 

blages 

Taxonomy of Buzzards Bay benthic communities 
The trophic structure of benthic communities I and II 
Sedimentology for ecologists 
Evolutionary and ecologic significance of oxygen-poor 

environments 
The evolution of benthic communities, pre-Cambrian to 

recent 

F. J. GRASSLE Benthic sampling 

Genetic variability of benthic communities 
H. L. SANDERS Biological effect of an oil spill 

E. O. WILSON Principles of biogeography I and II 

Principles of speciation I and 1 1 

Chemical communication I and II 

Principles of Sociobiology 

J. ATEMA Chemical communication III 

J. TODD An ethology of stress environment 

G. E. JONES The fate of freshwater bacteria in the sea 

The significance of heavy metals to marine bacteria 
W. VISHNIAC Productivity and exploitation of the ocean 

Planetary and biological evolution 
L. B. SLOBODKIN Ecology, a science, a movement, and a crisis 

NEUROBIOLOGY 

I. INSTRUCTORS 

MICHAEL V. L. BENNETT, Professor of Anatomy, Albert Einstein College of Medicine, 

Yeshiva University, co-director of course 
JOHN E. DOWLING, Associate Professor of Ophthalmology and Biophysics, Johns 

Hopkins University School of Medicine, co-director of course 
RODOLFO R. LLINAS, Associate Member, Education and Research, Foundation of the 

American Medical Association 
GEORGE PAPPAS, Professor of Anatomy, Albert Einstein College of Medicine, Yeshiva 

University 
FELIX STRUMWASSER, Professor in the Division of Biology, California Institute of 

Technology 
VICTOR WHITTAKER, Sir William Dunn Reader in Biochemistry, Cambridge University 

II. SPECIAL LECTURERS 

STEPHEN G. WAXMAN, Postdoctoral fellow, Albert Einstein College of Medicine 

GEORGE KATZ, Assistant Professor, Columbia University 

A. L. F. GORMAN, Research Physiologist, National Institute of Mental Health 

MAURIZIO MIROLLI, St. Elizabeth Hospital, Washington D. C. 

R. L. CHAPELL, Assistant Professor, Hunter College 

JOHN LISMAN, Massachusetts Institute of Technology 

CHARLES NICHOLSON, Assistant Professor, University of Iowa 

III. LECTURES 

GEORGE D. PAPPAS Nerve cells and the fine structure of membranes 

Structure and function of synapses 
STEPHEN G. WAXMAN Myelin and nodes of Ranvier 



30 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



M. Y. L. BENNETT 

GEORGE KATZ 
A. L. F. GORMAN 



MAURI/IO MIROLLI 
A. L. F. GORMAN 

J. E. DOWLIM; 



R. L. CHAPPELL 
J. E. DOWLIXG 
A. L. F. GORMAN 

M. V. L. BENNETT 



A. B. STEIN BACH 
M. V. L. BENNETT 

RODOLFO LUX AS 
JOHN LISMAN 
CHARLES NICHOLSON 
RODOLFO LLINAS 



Central dogma of neuro-physiology. I. Impulses 

Central dogma of neuro-physiology. II. Synapses 

Introduction of electrophysiological measurements 

Membrane theory: properties of nerve membrane 

Membrane potential of nerve and glial cells and diffusion 
through the extracellular space 

Geometrical factors determining the electrotonic properties 
of nerve cells 

Metabolism of nerve cells: Na-K exchange pump and 
electrogenic effects 

Specialized regions of nerve cells: Synaptic and sensory 
membrane 

Introduction to the visual system: Anatomy, chemistry, 
receptor potentials 

The lateral eye of Limulus 

Synaptic activation by receptor slow potentials 

The processing of visual information 

Comparative physiology of hyperpolarizing photoreceptor 
potentials 

lontophoretic application of drugs and micropharmacology 

Electric organs: comparative physiology and experimental 
utility 

Transmission at synapses of electroreceptors 

Functions of electrotonic synapses 

Interpretation of intracellular recordings in the CNS 

Cerebellar electrophysiology as a model for the analysis 
of central neuronal networks 

Limulus photoreceptors : The role of Ca+ in light- 
adaptation 

Analysis of field potentials evoked by populations of 
spatially oriented central neurons 

Dynamic properties of the responses of cerebrellar neu- 
ronal circuits to natural stimuli 



SYSTEMATICS-ECOLOGY PROGRA.A I 

THE STAFF 

Director: MELBOURNE R. CARRIKER 
Resident Systematist (Zoology) : ARTHUR G. HUMES 
Acting Resident Systematist (Botany) : ROBERT T. WILCE 
Resident Ecologist : IVAN VALIELA 

Resident Environmental Physiologist: WILLIAM C. STEWART 
Curator (Zoology) : JOHANNA M. REINHART 
Curator (Botany, part time) : WESLEY N. TIFFNEY 

Postdoctoral Fellows and Research Associates: JAMES FIORE, RAYMOND P. MARKEL, 
LAWRENCE R. MCCLOSKEY, ALLAN D. MICHAEL, LELAND W. POLLOCK, WILLIAM 

J. WOELKERLING 

Graduate Research Trainees: JOHN ALDRICH, CHARLENE D'AVANZO, BRUCE W. FOUND, 
WALTER HATCH, STEWART JACOBSON, WARREN KAPLAN, CHARLES KREBS, JOE 
WRIGHT, ROY M. YARNELL 

Yisiting Investigators: MARIE B. ABBOTT, LOUISE BUSH, PATRICIA L. DUDLEY, JOEL 
S. O'CONNOR, PHILIP PERSON, HAROLD H. PLOUGH, WESLEY N. TIFFNEY, RUTH 
D. TURNER, WILLIAM C. SUMMERS, WILLIAM J. WOELKERLING, DAVID K. YOUNG 



REPORT OF THE DIRECTOR 



31 



Collaborators: DAVID DOWNING, VIRGINIA PETERS, JAMES SCHAADT 

Visitors: DONALD BOURNE, KATHARINE D. HOBSON 

Consultants: WILLIAM RANDOLPH TAYLOR, RUTH D. TURXEK, ROBERT T. WILCE 

Administrative Assistant: CONSTANCE A. BRACKETT 

Program Secretary: EVA S. MONTIERO 

Technical Field Assistant: PETER J. OLDHAM 

Scientific Illustrators: RUTH VON ARX, SUSAN P. HELLER 

Captains, R/V A. E. VERRILL: PETER GRAHAM, FRANCIS J. DOOHAX 

Mates: FRANCIS J. DOOHAN, STEPHEX K. YOUNG 

Boatswain: WILLIAM PLETTXER 

Research Assistants: ANN CODY, GENE CROUCH, THEODORE J. GRANT (Bibliographer), 
DUDLEY L. GREELEY, PATRICIA HUGHES, TRACY MCL,ELLAN, JOHN J. MCMAHON, 
NANCY McNELLY, MARGARET A. MILLS (Biotic Census Project Ass't), HELEN 
ORTINS, CAROL PARMENTER, PAUL V. RUHLMAN, GREG RUPPERT, WARREN SASS 
(computer operator), CAROL Q. SCHWAMB, MARTHA SWARTZ, ERIC TEAL, RICHARD 
J. TRAVERSE, DIRK VANZAXDT, MARY LEE P. WILSON, RICHARD WYNNE 

Participants: NELL BACKUS, SHIRLEY HOLBROOK 



ROY M. YARNELL 
POUL HEEGAARD 

HAROLD H. PLOUGH 
ELDON BALL 
EDWARD CARPENTER 

JOEL S. O'CONNOR 
WILLIAM C. STEWART 

ROBERT BLUMBERG 

DONALD J. ZINX 
PAUL E. HARGRAVES 

JOHX C. HATHAWAY 



LAWREXCE R. MCCLOSKEY 
HERBERT W. GRAHAM 
DENNIS POLIS and 

DON MAURER 
GEORGE P. FULTON 

JAMES D. LAZELL 

HEXRY CAMPBELL 
RANDALL B. FAIRBANKS 

BRIAN D. BORNHOLD and 

JOHN D. MILLIMAN 
WILLARD D. HARTMAX 
MARVIN C. MEYER 



SEP SEMINARS (WIXTER IXCLUDED) 

What I am doing in the salt marsh 

Ovarial structures in the penaeids and larval stages and 

growth in the decapods 
Distribution changes in ascidian species on the continental 

shelf: evolution in fifty years 
Electrical correlates in behavior in the solitary hydroid, 

Corymorpha pal ma 

Distribution of diatoms on pelagic Sargassum and utiliza- 
tion of urea by some marine phytoplankters 
Benthic invertebrates of Moriches Bay, Long Island 
A study of the nature of the attractant in the Ophiodromus 

pngettensis-Patiria lineata commensal association 
Legal problems relating to the oceanic environment in 

Massachusetts 

Exploitation of the Alaskan Tundra 
Observations on phytbplankton of Narragansett Bay, 

Rhode Island 
Composition and movement of fine grained sediments 

along the Atlantic Coast 

Tektite II: impressions, decompressions, and digressions 
What happened to BCF? 
Planning for the baseline study of the Delaware Bay 



Trends in microvascular research (illustrated by cinephoto- 
microscopy) 

Herpetology of Cape Cod and the Islands: problems of 
distribution 

The role of the Coast Guard in water pollution 

An assessment of the effects of power generation upon the 
Cape Cod Canal sport fishery 

Phylogenetic and environmental influences upon the com- 
position of serpulid (Polychaeta) tubes 

Some living fossils among the sponges 

Taxonomy of marine leeches in retrospect and prospect 



32 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



MARIE B. ABBOTT 
JOHN H. DEARBORN 
RICHARD C. KUGLER 

ARTHUR G. HUMES 
PAUL TESSIER 

EDWARD GILFILLAN 



ELLSWORTH WHEELER 
PAUL GODFREY 

BRUCE COULL 
ALLAN D. MICHAEL 

HENRY M. REISWIG 
KENNETH TURGEON 
JOHN FIELD 
H. P. JEFFRIES 

RICHARD L. MILLER 
H. BURR STEINBACH 

BERTON ROFFMAN 
ROLAND L. WIGLEY 
RICHARD HENNEMUTH 

JAMES PARMENTIER 

OSCAR Liu 

P'RANK BELAMARICH 

ROBERT LIVINGSTONE 

JOHN LEE 

ROBERT K. SELANDER 
R. W. CASTENHOLZ 
WILLIAM COOPER 

SUMNER RlCHMAN 



Bryozoan populations of Block Island Sound 

Ecological studies of polar echinoderms 

Whaling under sail: some aspects of the American 

experience 

Copepods associated with marine invertebrates 
Biological adaptations of marine sand microfauna ; and 

Halamohydra (film) 
Physiological changes occurring during the invasion of the 

coastal environment by an oceanic species of zooplank- 

iin, Eitpliaitsia pacifica Hansen 
On the ecology of carnivorous Copepoda 
Ecological implications of oceanic overwash : dunes, dikes, 

and enginerr> 

Shallow water meiobenthos of the Bermuda platform 
Environmental factors and species distribution in Cape 

Cod Bay 
Comparative physiology of field populations of Jamaican 

Demospongiae 
The effects of cornstarch and dextrose supplements on 

oysters 
Cluster analysis: a tool for studying marine benthic 

communities 
Phytoplankton-zooplankton relationships: aspects of a 

biochemical enigma 
Chemotaxis of coelenterate sperm 
The Woods Hole Oceanographic Institution educational 

program 
Algal symbiosis 

Biology of the northern shrimp 
Exploitation and management of fishery resources of the 

northwest Atlantic 
Analysis of glutamic acid receptor sites in gastropod 

neurons 

Effects of chlorination on viruses in water 
Aggregation of amoebocytes in Limiiliis 
Haddock spawning and maturity studies past and 

present 
The microbiology of 1 cc of a salt marsh epiphytic 

community 

Systematic applications of the allozyme technique 
Species interaction in a hot spring 
Recent views on systems analysis of ecosystems 
Selective feeding behavior of Woods Hole copepods 



THE LABORATORY STAFF 
HOMER P. SMITH, GENERAL MANAGER 

Miss JANE FESSENDEN, Librarian 

JOHN J. VALOIS, Manager, Supply De- 
partment 

FRANK A. WILDES, Controller 

ROBERT KAHLER, Superintendent Build- 
ings and Grounds 



ROBERT GUNNING, Assistant Superinten- 
dent, Buildings and Grounds 

ROBERT B. MILLS, Manager, Department 
of Research Service 



REPORT OF THE DIRECTOR 



33 



GENERAL OFFICE 



Miss SUSAN M. BARNES 
EDWARD J. BENDER 
MRS. FLORENCE S. BUTZ 
MRS. VIVIAN I. MANSON 



Miss ELAINE C. PERRY 
MRS. CYNTHIA S. REGAN 
Miss MARY TAVARES 
MRS. ELIZABETH L. WHITE 



LIBRARY 



MRS. VIRGINIA BRANDENBURG 
DAVID J. FITZGERALD 
Miss MICHELLE T. BOSCH 



MRS. LENORA JOSEPH 
MRS. DORIS T. RICKER 



MAINTENANCE OF BUILDINGS & GROUNDS 



ELDON P. ALLEN 
LEE BOURGOIN 
BERNARD F. CAVANAUGH 
JAMES S. CLARKE 
CECIL COSTA 
Miss ALMA DAVIS 
JOHN V. DAY 
MANUEL P. DUTRA 
CHARLES FUGLISTER 
RICHARD E. GEGGATT, JR. 
MRS. ELIZABETH KUIL 



DONALD B. LEHY 
RALPH H. LEWIS 
RUSSELL F. LEWIS 
WILSON LITTLE 
MICHAEL W. LOVE 
ALAN G. LUNN 
ANTHONY J. MAHLER 
STEPHEN MILLS 
FREDERICK E. THRASHER 
FREDERICK E. WARD 
RALPH WHITMAN 



DEPARTMENT OF RESEARCH SERVICE 



GAIL M. CAVANAUGH 
JIM A. HANCOCK 
LOWELL V. MARTIN 



Miss CHRISTINE A. SIMMONS 
FRANK E. SYLVIA 



BRADFORD F. ELLIS 
EDWARD ENOS 
DAVID H. GRAHAM 
LEWIS M. LAWDAY 
ROBERT O. LEHY 



SUPPLY DEPARTMENT 

Miss JOYCE B. LIMA 
EUGENE TASSINARI 
BRUNO F. TRAPASSO 
JOHN YARAO 



3. INVESTIGATORS: LILLIE, GRASS, AND RAND FELLOWS; STUDENTS 

Independent Investigators, 1971 

ACHK, BARRY W., Assistant Professor of Zoology, Florida Atlantic University 

ADELMAN, WILLIAM J., Professor of Physiology, University of Maryland 

ALBUQUERQUE, EDSON X., Associate Professor of Pharmacology, State University of New York 

at Buffalo 

ANDERSON, EVERETT, Professor of Zoology, University of Massachusetts 
ANDREWS, THOMAS G., JR., Investigator, Columbia University 

APLEY, MARTYN L., Assistant Professor of Biology, Brooklyn College, City University of New York 
ARISPE, NELSON, Research Associate, Duke University 
ARMSTRONG, CLAY M., Associate Professor of Physiology, University of Rochester 



34 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

ARMSTRONG, PHILIP B., Professor Emeritus, State University of New York, Upstate Medical 

Center 

ARNOLD, JOHN M., Associate Professor, University of Hawaii 
BARLOW, ROBERT B., JR., Associate Professor, Syracuse University 
BARNES, ANTHONY T., Postdoctoral Fellow, University of California, Santa Barbara 
BARTELL, CLELMER K., Assistant Professor, Louisiana State University 
EARTH, LESTER, Independent Investigator, Marine Biological Laboratory 
BARTH, LUCENA, Independent Investigator, Marine Biological Laboratory 
BEAUCHAMP, Ross, Postdoctoral Fellow, Washington University Medical School 
BELAMARICH, FRANK A., Associate Professor of Biology, Boston University 
BELL, ALLEN L., Assistant Professor of Anatomy, University of Colorado Medical Center 
BENNETT, MICHAEL Y. L., Professor of Anatomy, Albert Einstein College of Medicine 
BERMAN, MAC, Research Assistant, Columbia LTniversity 
BETCHAKU, TEIICHI, Instructor of Biology and Director of Laboratory for Biological Ultrastruc- 

ture, Yale LTniversity 

BEZANILLA, FRANCISCO, Postdoctoral fellow and Associate in Physiology, University of Rochester 
BITO, LASZLO Z., Assistant Professor of Ophthalmology, Columbia University 
BLINKS, JOHN R., Head, Department of Pharmacology, Mayo Foundation 
BORGESE, THOMAS A., Assistant Professor of Biology, Lehman College, City University of New 

York 

BORN, GUSTAV V. R., Vandervell Professor of Pharmacology, University of London 
BOURNE, DONALD W., Research Biologist, Marine Research Foundation, Inc. 
BRANDT, PHILIP W., Associate Professor, Columbia University, College of Physicians and Surgeons 
BRAUN, ANDREW G., Postdoctoral Fellow, Brandeis University 
BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University 
BROWN, JOEL E., Associate Professor of Physiology, Massachusetts Institute of Technology 
BURDICK, CAROLYN J., Assistant Professor, Brooklyn College, City University of New York 
BURGER, MAX M., Associate Professor of Biology and Biochemical Sciences, Princeton University 
CAMHI, JEFFREY M., Assistant Professor of Biology, Cornell University 
CARBONE, EMILIO, Yisiting Fellow, National Institute of Mental Health 
CASE, JAMES F., Professor, University of California, Santa Barbara 
CASTENHOLZ, RICHARD \Y., Professor of Biology, University of Oregon 
CERVETTO, LUIGI, Public Health Service International Fellow, National Institute of Neurological 

Diseases and Stroke 

CHAMBERLAIN, JACK K., Research Fellow in Hematology, University of Rochester School of Medi- 
cine and Dentistry 

CHAPMAN, GARTH, Professor of Zoology, Queen Elizabeth College, University of London 
CHAPPELL, RICHARD L., Assistant Professor, Hunter College, City University of New York 
CLAYTON, RODERICK K., Professor, Cornell University 
CLEMENT, ANTHONY C., Professor of Biology, Emory University 
COHEN, ADOLPH L, Professor of Anatomy, Research Professor of Ophthalmology, \Yashington 

University Medical School 

COHEN, LAWRENCE B., Assistant Professor, Yale University School of Medicine 
COLE, KENNETH S., Senior Research Biophysicist, NINDS, National Institutes of Health 
COLLIER, J. R., Professor of Biology, Brooklyn College, City University of New York 
COLLINS, MICHAEL F., Assistant Professor of Zoology, University of Texas at Austin 
COLWIN, ARTHUR L., Professor of Biology, Queens College, City L T niversity of New York 
COLWIN, LAURA HUNTER, Professor of Biology, Queens College, City University of New York 
CONNOR, JOHN A., Assistant Professor of Physiology, University of Illinois 
COOPERSTEIN, SHERWIN J., Professor of Anatomy, University of Connecticut 
CORNELL, NEAL W., Associate Professor of Chemistry, Pomona College 
COSTELLO, DONALD PAUL, Kenan Professor of Zoology, University of North Carolina 
COUSINEAU, GILLES H., Associate Professor, Universite de Montreal 
CRIPPA, MARCO, Professor, CNR Laboratory of Molecular Embryology, Napoli, Italy 
DAVIDSON, ERIC H., Associate Professor, California Institute of Technology 
DAVILA, HECTOR, Research Fellow, Yale University 

DAVIS, WILLIAM J., Assistant Professor of Biology, University of California, Santa Cruz 
DAW, NIGEL W., Assistant Professor, Washington University Medical School 
DEGROOF, ROBERT C., Graduate student, Duke LTniversity 



J 
REPORT OF THE DIRECTOR 35 

DELEZE, JEAN-BERNARD, Visiting Associate Professor of Physiology, Columbia University College 

of Physicians and Surgeons 
DELORENZO, A. J. DARIN, Director of Research, Department of Surgery, The Johns Hopkins 

University School of Medicine 

DEpHiLLiPS, HENRY A., JR., Associate Professor of Chemistry, Trinity College 
DETERRA, NOEL, Assistant Member, The Institute for Cancer Research 

DETWILER, PETER BENTON, Staff Fellow, National Institute of Neurological Diseases and Stroke 
DIMOCK, RONALD V., Assistant Professor of Biology, Wake Forest University 
DODGE, FREDERICK A., JR., Adjunct Associate Professor, The Rockefeller University 
DOWDALL, MICHAEL J., Senior Assistant in Research, University of Cambridge, Cambridge, 

England 

DOWLING, JOHN E., Associate Professor, Johns Hopkins University 
DRISCOLL, EGBERT G., Professor, Wayne State University 
DUNHAM, PHILIP B., Professor of Physiology, Syracuse University 
EASTWOOD, ABRAHAM, III., Trainee Fellow, Columbia University 
EDYUD, LASZLO G., Co-Director of Research, Institute of Muscle Research at the Marine Biological 

Laboratory 

EHRENSTEIN, GERALD, Research Physicist, National Institutes of Health 
ERULKAR, S. D., Professor of Pharmacology, University of Pennsylvania 
FARMANFARMAIAN, A., Associate Professor of Physiology, Rutgers University 
FERTZIGER, ALLEN PHILIP, Assistant Professor, University of Maryland 
FINCK, HENRY, Associate Professor of Anatomy and Cell Biology, University of Pittsburgh School 

of Medicine 

FINE, JACOB, Director of Shock Division, Harvard Surgical Unit, Boston City Hospital 
FINGERMAN, MILTON, Professor of Biology, Tulane University 
FINN, ARTHUR LEONARD, Associate Professor of Medicine, University of North Carolina, School 

of Medicine 
FISHMAN, HARVEY M., Assistant Professor of Biological Sciences, State University of New York 

at Albany 

Fox, MAURICE S., Professor of Genetics, Massachusetts Institute of Technology 
FRANKE, J., Research Assistant, Brandeis University 
FRAZIER, DONALD T., Associate Professor, University of Kentucky 
FREEMAN, GARY, Assistant Professor of Biology, University of California, San Diego 
FUORTES, M. G. F., Chief, Laboratory of Neurophysiology, National Institute of Neurological 

Diseases and Stroke 

GELPERIN, ALAN, Assistant Professor of Biology, Princeton University 
GIBBS, MARTIN, Professor of Biology, Brandeis University 
GILBERT, DANIEL L., Head, Section on Cellular Biophysics, National Institute of Neurological 

Diseases and Stroke 

GOLDMAN, MARVIN B., Neurologist, Toronto 

GOOCH, JAMES L., Assistant Professor of Biology, Juniata College 

GORDON, ALLEN R., Postdoctoral Fellow, Columbia University, College of Physicians and Surgeons 
GORMAN, ANTHONY L. F., Research Physiologist, St. Elizabeth Hospital 
GRANT, DAVID C., Assistant Professor of Biology, Davidson College 
GRANT, PHILIP, Professor of Biology, University of Oregon 

GREENBERG, MICHAEL JOHN, Associate Professor of Biological Science, Florida State University 
GROSCH, DANIEL S., Professor of Genetics, North Carolina State University at Raleigh 
GROSSMAN, ALBERT, Associate Professor, New York University Medical Center 
GROSSMAN, LAWRENCE, Professor of Biochemistry, Brandeis University 
GRUNDFEST, HARRY, Professor of Neurology, Columbia University, College of Physicians and 

Surgeons 

GUERRIER, P., University of Montreal 

GUILLARD, ROBERT R. L., Associate Scientist, Woods Hole Oceanographic Institution 
GUTTMAN, RITA, Professor of Biology, Brooklyn College, City University of New York 
HALLETT, MARK, Staff Associate, National Institute of Mental Health 
HALVORSON, HARLYN O., Professor of Molecular Biology and Bacteriology, University of 

Wisconsin 
HARDING, CLIFFORD V., Professor and Chairman, Department of Biological Sciences, Oakland 

University 



36 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

HARRIS, EDWARD M., Electronic Engineer, Duke University 

HASCHEMEYER, AUDREY E. V., Associate Professor of Biological Sciences, Hunter College, City 

University of New York 

HASELKORN, ROBERT, Professor and Chairman, Department of Biophysics, University of Chicago 
HAYES, RAYMOND L., Associate Professor of Anatomy and Cell Biology, University of Pittsburgh 
HEIDGER, PAUL M., JR., Assistant Professor of Anatomy, Tulane University 
HENLEY, CATHERINE, Research Associate, University of North Carolina 
HEUSER, JOHN E., Research Associate, National Institutes of Health 
HILDEN, SHIRLEY, Assistant Professor, University of Idaho 
HILL, ROBERT B., Associate Professor, Lhiiversity of Rhode Island 
HINEGARDNER, RALPH, Associate Professor, University of California, Santa Cruz 
HINSCH, GERTRUDE \V., Associate Professor, Institute of Molecular Evolution, University of 

Miami 

HOFFMAN, ALBERT C., Assistant Professor of Biology, Millersville State College 
HOLLYFIELD, JOE, Assistant Professor of Ophthalmology, Columbia University 
HOLZ, GEORGE G., Professor and Chairman, Department of Microbiology, State University of 

New York, Upstate Medical Center 

HOSKIN, FRANCIS C. G., Professor of Biology, Illinois Institute of Technology 
HUBBARD, RUTH, Research Associate and Lecturer, Harvard University 
HUMMON, WILLIAM D., Assistant Professor of Zoology, Ohio University 
HUXLEY, HUGH E., Scientific Staff, M. R. C. Laboratory of Molecular Biology, Cambridge, 

England 

ILAN, JOSEPH, Associate Professor, Temple University 
INGE, WELLFORD W., Staff Surgeon, Brooke Army Medical Center 
INOUE, SADAYUKI, Assistant Professor, L T niv<TMty of Montreal 
JANNASCH, HOLGER W., Senior Scientist, Woods Hole Oceanographic Institution 
JENSEN, ARNE, Research Scientist, University of Trondheim, Norway 
JENSEN, SYNN^VE LIAAEN, Professor, Norwegian Institute of Technology, Norway 
JENNINGS, JOSEPH BRIAN, Reader in Invertebrate Zoology, University of Leeds, England 
JOHNSON, MICHAEL L., Instructor, University of Connecticut 

JOHNSON, WALTER ERWIN, Welch Foundation Postdoctoral Fellow, University of Texas at Austin 
JOSEPHSON, ROBERT K., Professor of Biology, Case Western Reserve University 
KALTENBACH, JANE C., Professor, Mount Holyoke College 

KAMINER, BENJAMIN, Chairman and Professor, Boston University School of Medicine 
KATZ, GEORGE M., Assistant Professor, Columbia University 

KAWAI, NOBUFUMI, Research Associate, Columbia University, College of Physicians and Surgeons 
KEAN, EDWARD L., Assistant Professor of Biochemistry and Ophthalmology, Case Western Reserve 

University 

KELLY, ROBERT E., Assistant Professor, Dartmouth Medical School 
KITZ, RICHARD J., Henry Isaiah Dorr Professor, Harvard Medical School and Anesthetist-in- 

Chief, Massachusetts General Hospital 
KORN, HENRI, Research Associate, Albert Einstein College of Medicine and Faculty of Medicine, 

Paris, France 
KRINSKY, NORMAN L, Professor of Biochemistry and Pharmacology, Tufts University School of 

Medicine 

KRUPA, PAUL L., Associate Professor, The City College of the City University of New York 
KUHNS, WILLIAM J., Associate Professor of Pathology, New York University School of Medicine 
KUSANO, KIYOSHI, Associate Professor, Illinois Institute of Technology 

LARSEN, WILLIAM J., Research Associate, Columbia University, College of Physicians and Surgeons 
LASER, RAYMOND J., Assistant Professor, Case Western Reserve L T niversity 
LASH, JAMES W., Professor of Anatomy, L T niversity of Pennsylvania, School of Medicine 
LATORRE, RAMON, Visiting Fellow, National Institutes of Health 
LAUFER, HANS, Associate Professor of Biology, University of Connecticut 
LAZAROW, ARNOLD, Professor and Head, Department of Anatomy, University of Minnesota 
LECAR, HAROLD, Physicist, National Institutes of Health 
LEE, JOHN J., Associate Professor and Chairman, Graduate Studies Committee, City College of 

the City University of New York 

LERMAN, SIDNEY, Professor of Ophthalogy and Biochemistry and Directory, Department of Ex- 
perimental Ophthalmology, McGill University 



REPORT OF THE DIRECTOR 

LEVIN, JACK, Associate Professor of Medicine, Johns Hopkins University School of Medicine 
LEVINTHAL, CYRUS, Professor of Biology, Columbia University 

LEVY, MILTON, Professor of Biochemistry, New York University, College of Dentistry 
LINDBERG, ROBERT B., Chief, Microbiology Branch, U. S. Army Institute of Surgical Resean h 
LIPICKY, RAYMOND JOHN, Assistant Professor of Pharmacology and Medicine, University of 

Cincinnati 

LIUZZI, ANTHONY, Assistant Professor, Yale University 
LIVENGOOD, DAVID R., Postdoctoral Fellow, Institute of Psychiatric Research, Indiana University 

Medical Center 

LLINAS, R., Professor, Department of Physiology, University of Iowa 
LOEWENSTEIN, WERNER, Professor Physiology, Columbia University, College of Physicians and 

Surgeons 

LOEWUS, FRANK A., Professor, State University of New York at Buffalo 
LOFTFIELD, ROBERT B., Chairman, Department of Biochemistry, University of New Mexico 

School of Medicine 

LONGO, FRANK J., Assistant Professor, University of Tennessee Medical Units 
LOKAND, L., Professor of Chemistry, Northwestern University 
LUCAS, ROGER C., Postdoctoral Fellow, Brandeis L T niversity 
MACNICHOL, EDWARD F., JR., Director, National Institute of Neurological Diseases and Stroke, 

National Institutes of Health 

MAGUN, BRUCE E., Assistant Professor of Anatomy, University of Tennessee 
MAHLER, HENRY R., Research Professor of Chemistry, Indiana University 
MANALIS, RICHARD S., Assistant Professor of Physiology, University of Cincinnati 
MANGUM, CHARLOTTE PRESTON, Associate Professor of Biology, College of William and Mary 
MATSUMURA, FUMIO, Associate Professor, University of Wisconsin 
McAFEE, DONALD A., Postdoctoral Fellow, Yale University School of Medicine 
McREYNOLDS, JOHN S., Staff Associate, National Institute of Neurological Diseases and Stroke, 

National Institutes of Health 

MENDELSON, MARTIN, Associate Professor of Physiology, New York l'niversity School of Medicine 
METUZALS, J., Professor, University of Ottawa 
METZ, CHARLES B., Professor, University of Miami 
MILKMAN, ROGER, Professor of Zoology, The University of Iowa 
MILLER, JAMES A., JR., Chairman, Department of Anatomy, Tulane University 
MILLER, RICHARD L., Assistant Professor of Biology, Temple University 
MINOR, RONALD R., Postdoctoral in Pathology, University of Pennsylvania 
MITCHELL, RALPH, Gordon McKay Professor of Applied Biology, Harvard University 
MOORE, JOHN W., Professor of Physiology, Duke University 
MOORE, NATALIE B., Graduate Student, University of North Carolina 
MORIN, JAMES G., Assistant Professor of Zoology, University of California, Los Angeles 
MOTE, MICHAEL L, Assistant Professor of Biology, Temple University 
MURER, ERIK HOMANN, Research Fellow at the University of Oslo 
NAKA, KEN-!CHI, Research Associate, California Institute of Technology 
NARAHASHI, TOSHIO, Professor and Head of Pharmacology Division, Duke l'niversity 
NELSON, LEONARD, Professor and Chairman, Department of Physiology, Medical College of Ohio 

at Toledo 
NEWELL, PETER C., CRC Fellow and Lecturer in Microbiology at St. Peter's College, University 

of Oxford, England 

NICHOLSON, C., Assistant Professor, University of Iowa 
NOLTE, JOHN, Postdoctoral Fellow, University of Colorado Medical School 
NYSTROM, RICHARD A., Associate Professor, University of Delaware 

O'BRIEN, ELINOR M., Lecturer, Associate Director, Cancer Research Institute of Boston College 
OHKI, SHINPEI, Assistant Professor of Pharmaceutics and Biophysics, State University of New 

York at Buffalo 

OHTA, MASAHIRO, Postdoctoral Research Associate, Duke University 
O'MELIA, ANNE F., Research Fellow in Biological Chemistry, Harvard Medical School 
OOSTING, PIETER H., Scientist, Philips Research Laboratory, Holland 
OKENTLICHER, MORTON D., NINDS Special Research Fellow, Columbia University 
OSMAN, RICHARD W., Graduate Student, University of Chicago 
PAGE, CHARLES H., Assistant Professor of Zoology, Ohio University 



38 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

PALTI, YORAM, Chairman, Department of Physiology, Hebrew University Medical School and The 
Haifa Medical School 

PAPPAS, GEORGE D., Professor of Anatomy and Deputy Chairman of Department of Anatomy, 
Albert Einstein College of Medicine 

PAUL, DOROTHY H., Postdoctoral Research Fellow, Tufts University 

PEARLMAN, ALAN L., Assistant Professor of Physiology and Neurology, Washington University 
School of Medicine 

PFOHL, RONALD J., Assistant Professor, Miami University 

PIERCE, SIDNEY K., JR., Assistant Professor, University of Maryland 

PIKO, LAJOS, Chief, Developmental Biology Laboratory, Veterans Hospital, Sepulveda, California 

PITMAN, ROBERT M., Research Staff Biologist, Yale University 

PODOLSKY, RICHARD J., Chief, Section on Cellular Physics, National Institutes of Health 

POLITOFF, ALBERTO, Interdepartmental Research Fellow, Albert Einstein College of Medicine 

PRENDERGAST, ROBERT A., Associate Professor of Ophthalmology and Pathology, The Johns 
Hopkins University School of Medicine 

PRICE, C. A., Professor of Plant Biochemistry, Rutgers UJniversity 

PRZYBYLSKI, RONALD J., Assistant Professor, Case Western Reserve University 

RAFF, MARTIN C., Visiting Scientist, National Institute for Medical Research, London 

RAMON, FIDEL, Research Associate, Duke University 

RAMSEY, W. SCOTT, Postdoctoral Research Fellow, Yale University 

RAO, BALAKRISHNA R., Associate Professor, East Stroudsburg State College 

RAO, K. RANGA, Research Associate, Tulane UJniversity 

REDDAN, JOHN R., Associate Professor and Associate Chairman, Department of Biological Sci- 
ences, Oakland University 

REESE, THOMAS S., Head, Section on Functional Neuroanatomy, National Institute of Neurologi- 
cal Diseases and Stroke 

REUBEN, JOHN P. Associate Professor, Columbia University, College of Physicians and Surgeons 

REYNOLDS, GEORGE T., Professor of Physics, Princeton University 

RHOADS, DONALD C., Associate Professor, Yale University 

RICE, ROBERT V., Professor of Biochemistry and Head, Department of Biological Sciences, 
Carnegie-Mellon University 

RICHMAN, SUMNER, Professor of Biology, Lawrence University 

RIPPS, HARRIS, Professor, New York University School of Medicine 

RITCHIE, J. MURDOCH, Chairman, Department of Pharmacology, Yale UJniversity School of 
Medicine 

ROBINSON, KENNETH R., Graduate Student, Purdue University 

ROSE, BIRGIT, Research Associate, Columbia University College of Physicians and Surgeons 

ROSE, S. MERYL, Professor, Tulane University 

ROSENBLUTH, JACK, Associate Professor of Physiology, New York University College of Medicine 

RUSHFORTH, NORMAN B., Associate Professor of Biology, Case Western Reserve University 

RUSSELL-HUNTER, W. D., Professor of Zoology, Syracuse University 

RUSTAD, RONALD C., Associate Professor of Radiology and Biology, Case Western Reserve 
University 

SAN PIETRO, ANTHONY', Professor and Chairman, Botany Department, Indiana University 

SAUNDERS, JOHN W., JR., Professor of Biological Sciences, State University of New York at Albany 

SCHIFF, JEROME A., Professor of Biology, Brandeis University 

SCHNEIDER, ALLAN, Staff Fellow, National Institute of Mental Health 

SCHOEPF, CLAUDE, Illustrator-Photographer, Columbia University 

SCHOPF, THOMAS J. M., Assistant Professor, University of Chicago 

SCHRAMECK, JOAN E., Graduate Student, Stanford University 

SCHUEL, HERBERT, Assistant Professor, City University of New York, Mount Sinai School of 
Medicine 

SCHUETZ, ALLEN \V., Assistant Professor, Johns Hopkins University 

SCOTT, GEORGE T., Professor of Biology, Oberlin College 

SELANDER, ROBERT K., Professor of Zoology, LTniversity of Texas at Austin 

SENFT, JOSEPH PHILIP, Assistant Professor, Rutgers-The State University 

SEVCIK, CARLOS, Postdoctoral Fellow, Duke University Medical Center 

SEYAMA, ISSEI, Postdoctoral Research Associate, Duke University 

SHAPIRO, BERT I., Assistant Professor of Biology, Harvard University 



REPORT OF THE DIRECTOR 39 

SHEPKO, DAVID, Professor of Biology and Associate Professor of Surgery, Boston University 

SHERMAN, IRWIN W., Professor of Zoology, University of California, Riverside 

SHRIVASTAV, BRIJ BHUSHAN, Postdoctoral Fellow, Yale University 

SHULMAN, MARC J., Staff Fellow, National Cancer Institute, National Institutes of Health 

SIEGEL, IRWIN M., Associate Professor Experimental Ophthalmology, New York University 

Medical Center 

SILVERSTEIN, ARTHUR M., Professor, Johns Hopkins University School of Medicine 
SIMON, ERIC J., Associate Professor, New York University Medical School 
SKINNER, DOROTHY M., Staff member, Oak Ridge National Laboratory 
SMITH, GERALD N., JR., Assistant Professor of Zoology, University of Toronto 
SOIFER, DAVID, Senior Research Scientist, Institute for Research in Mental Retardation 
SORENSON, ALBERT LEE, Assistant Professor, Brooklyn College 
SORENSON, MARTHA M. Postdoctoral Trainee, Columbia University, College of Physicians and 

Surgeons 

SORENSON, ROBERT L., Assistant Professor, University of Minnesota 
SPIRA, MICHA E., Investigator, Albert Einstein College of Medicine 
SPRAY, DAVID C., Graduate Student, University of Florida 

SQUADRONI, JOSE, REV. S. J., Professor and Investigator, Colegio del Sagrado Corazon 
STAFFORD, WALTER, F., Ill, Graduate Student, University of Connecticut 
STEINBACH, ALAN B., Assistant Professor, University of California, Berkeley 
STEINBERG, MALCOLM S., Professor of Biology, Princeton University 
STEINBERG, SIDNEY, Research Associate, Columbia University 

STELL, WILLIAM K., Senior Staff Fellow, National Institute of Neurological Diseases and Stroke 
STEPHENS, RAYMOND E. Associate Professor of Biology, Brandeis University 

STRACHER, ALFRED, Professor and Acting Chairman, Department of Biochemistry, State Univer- 
sity of New York, Downstate Medical Center 

STUNKARD, HORACE W., Research Associate, American Museum of Natural History 
SULLIVAN, REV. WM. D., Professor/Director, Cancer Research Institute of Boston College, Boston 

College 

SUMMERS, ROBERT, Postdoctoral Fellow, Tulane University and University of Maine 
SUZUKI, JIRO, Research Associate, Columbia University, College of Physicians and Surgeons 
SZENT-GYORGYI, ALBERT, Director and Principal Investigator, Institute for Muscle Research, 

Marine Biological Laboratory 

SZENT-GYORGYI, ANDREW G., Professor of Biology, Brandeis University 
TAKASHIMA, SHIRO, Associate Professor, University of Pennsylvania 
TANNENBAUM, ALICE SUSAN, Trainee in Cytology, New York University Medical Center 
TASAKI, ICHIJI, Chief, Laboratory of Neurobiology, National Institute of Mental Health 
TAYLOR, ROBERT E., Acting Chief, Laboratory of Biophysics, National Institute of Neurological 

Diseases and Stroke 

TAYLOR, WM. RANDOLPH, Curator and Emeritus Professor of Botany, University of Michigan 
TELFER, WILLIAM H., Professor of Biology, University of Pennsylvania 
THOMAS, LEWIS, Professor and Chairman, Department of Pathology, Yale University School of 

Medicine 

TILNEY, LEWIS G., Associate Professor of Biology, University of Pennsylvania 
TRACER, WILLIAM, Professor, The Rockefeller University 

TRINKAUS, J. P., Professor of Biology and Master of Branford College, Yale University 
TROLL, WALTER, Professor, New York University Medical School 
TUPPER, JOSEPH T., Assistant Professor, Syracuse University 
TWEEDELL, KEN YON S., Professor of Biology, University of Notre Dame 
VAUGHN, JACK C., Associate Professor, Miami University 

VILLEE, CLAUDE A., Andelot Professor of Biological Chemistry, Harvard University 
VINCENT, WALTER S., Professor and Chairman, University of Delaware 
WAGNER, HENRY G., Director of Intramural Research, National Institute of Neurological Diseases 

and Stroke, National Institutes of Health 

WALD, GEORGE, Higgins Professor of Biology, Harvard University 

WALKER, MURIEL HELENA, Postdoctoral Research Associate, Institute of Molecular Evolution 
WANG, CHING MUH, Postdoctoral Research Associate, Duke University 
WATKINS, DUDLEY T., Assistant Professor, University of Connecticut Health Center 
WAXMAN, STEPHEN G., Postdoctoral Fellow, Albert Einstein College of Medicine 



40 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

WEBB, H. MARGUERITE, Professor of Biological Science, Goucher College 

WEBER, ANNEMARIE, Professor of Biochemistry, St. Louis University 

WEIDNER, EARL, Guest Investigator, The Rockefeller University 

WEIGHT, FORREST F., Chief, Section on Synaptic Pharmacology, National Institute of Mental 

Health 

WEISENBERG, RICHARD, Assistant Professor of Biology, Temple University 
WEISSMANN, GERALD, Professor of Medicine, New York University School of Medicine 
WHITTAKER, J. RICHARD, Associate Member, Wistar Institute of Anatomy and Biology 
WHITTAKER, V. P., Reader in Biochemistry, University of Cambridge, England 
\VIEDERHOLD, MICHAEL L., Staff Fellow, National Institute of Neurological Diseases and Stroke, 

National Institutes of Health 

WILSON, EDWARD O., Professor of Zoology, Harvard University 
WILSON, WALTER L., Professor, Oakland University 

WINE, JEFFREY J., Graduate student, University of California, Los Angeles 
WITKOVSKY, PAUL, Assistant Professor of Physiology, Columbia University College of Physicians 

and Surgeons 

WOLBARSHT, MYRON L., Professor of Ophthalmology, Duke University Medical Center 
WOLFE, RALPH S., Professor of Microbiology, University of Illinois 

WORTHINGTON, C. R., Professor of Chemistry and Physics, Mellon Institute of Science, Carnegie- 
Mellon University 

\\V, CHAU H., Postdoctoral Fellow, Duke L T niversity Medical Center 
WYNNE, MICHAEL J., Assistant Professor, LTniversity of Texas at Austin 
WYSE, GORDON A., Assistant Professor of Zoology, University of Massachusetts 
\\~YTTENBACH, CHARLES R., Associate Professor of Physiology and Cell Biology, University of 

Kansas 

YOUNG, JANICE E., Assistant Professor of Biology, Keuka College 
YPHANTIS, DAVID A., Professor of Biology, University of Connecticut 

/K.MAN, SEYMOUR, Associate Professor of Ophthalmology and Biochemistry, University of 
Rochester School of Medicine and Dentistry 

Lillie Fellow, 1971 

MARTIN C. RAFF, Visiting Scientist, National Institute for Medical Research, Mill Hill, London, 
England 

Grass Fellows, 1971 

FKAZIER, DONALD T., Senior Fellow, Associate Professor, University of Kentucky 
CONNOR, JOHN A., .Wistant Professor of Physiology and Biophysics, University of Illinois 
DAVIS, WILLIAM J., Assistant Professor of Biology, L T niversity of California, Santa Cruz 
DEGROOF, ROBERT C., Graduate Student, Duke University 
LASEK, RAYMOND J., Assistant Professor, Case Western Reserve University 

LIYENGOOD, DAVID R., Postdoctoral Fellow, Institute of Psychiatric Research, Indiana Univer- 
sity Medical Center 

PAGE, CHARLES H., Assistant Professor of Zoology, Ohio University 
PAUL, DOROTHY H., Postdoctoral Research Fellow, Tufts University 
PITMAN, ROBERT, Research Staff Biologist, Yale University 
SCHRAMECK, JOAN E., Graduate Student, Stanford University 
SPRAY, DAVID C., Graduate Student, University of Florida 
WINE, JEFFREY J., Graduate Student, University of California, Los Angeles 

Rand Fellow, 1971 
SELANDER, ROBERT K., Professor of Zoology, University of Texas at Austin 

Research Assistants, 1971 

ABBOTT, JANICE ELAYNE, National Institutes of Health 
ANTONELLIS, BLENDA CARLSSON, Case Western Reserve University 
AUGENFELD, JOHN M., University of Maryland, School of Medicine 
AVISE, JOHN C., University of Texas 
BANNER, JOHN L., Ill, Falmouth, Massachusetts 



KKPOKT OF THE DIRECTOR 41 

BARNES, STEPHEN N., University of Colorado Medical Center 

BEACH, DAVID H., State University of New York, Upstate Medical Center 

BELANGER, ANN M., Case Western Reserve University 

BELANGER, SANDRA E., The Biological Bulletin, Marine Biological Laboratory 

BELLER, DAVID, Princeton University 

BERGER, EDWARD M., University of Chicago 

Bi< IHOUSE, KATHY, Case Western Reserve University 

BOSCH, HERMAN F., The Johns Hopkins University 

BOWEN, RICHARD A., Rutgers University 

Box, SHARON, Mary C. Wheeler School 

BREHM, PAUL HARLAN, University of California, Los Angeles 

BRUNER, WILLIAM E., II, Case Western Reserve LTniversity 

BURROWS, ELIZABETH P., Temple University 

CAGAN, LAIRD, New York University Medical School 

CAMPBELL, LAURIE KATHERINE, Northwestern University 

CARBONETTO, SALVATORE, University of Massachusetts 

CARHART, JUDY ANN, College of William and Mary 

CARTER, JOSEPH G., Yale University 

CHIPKIN, ROBERT B., Union College 

CHOUINARD, ANNETTE, University of Montreal 

CHOW CHONG, PHILLIP, University of Ottawa 

CIANCI, LUIGI A., State University of \t-\v York, Downstate Medical Center 

CITKOWITZ, ELENA, Columbia University 

CLINTON, GAIL M., University of California, San Diego 

COLLIER, MARJORIE M., Brooklyn College of City University of New York 

CONNELL, MARGARET J., Florida State University 

COOPERSTEIN, LAWRENCE, Princeton University 

CUNNINGHAM, DENIS, Yale University 

DAVENPORT, JOHN E., University of New Mexico, School of Medicine 

DUBOIS, ROSAIRE, University of Montreal 

DULUDE, GAIL, National Institutes of Health 

DWYER, TERRY, University of Rochester Medical School 

ELLISON, REBECCA, Hunter College 

EVERSOLE, ARNOLD G., Syracuse University 

FAYBIK, KATHRYN, Columbia University 

FEIN, ALAN, Johns Hopkins University 

FKINGOLD, ROBERT E., Case Western Reserve University 

FINKEL, Lois, City University of New York 

FISHBURN, JOHN P. University of Iowa 

FISHER, JOHN BERTON, Yale University 

FOUNTAIN, GAIL, University of Massachusetts 

FRAIOLI, ANTHONY, Syracuse LTniversity 

FRIEDMAN, MARC, Brooklyn College 

FRIT/LER, MARVIN, LTniversity of Calgary 

GEPNER, IVAN, Princeton University 

GERSHWIN, RANDY JAY, Syracuse University 

GREBANIER, ALICE, Brooklyn College 

GRIFFIN, EVELYN M., Cornell University 

HABERFIELD, EVE, University of Rhode Island 

HAMILTON, DAVID P., University of Delaware 

HAUSE, SHELDON K., Illinois Institute of Technology 

HERVAS, ELOISE, McGill University 

HILL, MARGARET C., University of Kansas 

HOFFMAN, RUTH, Brandeis University 

Hoi. BROOK, SHIRLEY W., Boston University 

HUBERMAN, MICHAEL H., Trinity College 

HUNTER, VERNON DAVID, LTniversity of South Florida 

ILAN, JUDITH, Temple University 

JAMES, ALBERT, University of Connecticut 



42 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

JOHNSON, MICHAEL, University of Connecticut 

JOHNSON, SUSAN BAGLEY, University of Rochester 

KAHLER, GEORGE, Rice University 

KASHGARIAN, MICHAEL, Yale University 

KAUFMAN, KARL W., University of Chicago 

KAY, DOUGLASS A., University of Maryland Medical School 

KENNEDY, SAMUEL WATKINS, Tulane University 

KIMURA, JOHN E., Boston University School of Medicine 

KLEIN, NANCY C., University of Virginia 

KROPP, DONNA L., Syracuse University 

LANGER, GEORGE SCOTT, Lawrence University 

LANNI, CARMINE, Herbert Lehman College of City University of New York 

LEE, DAVID, Ottawa University 

LEETMAA, BONNIE L., University of Massachusetts 

LEHMAN, WILLIAM J., Brandeis University 

LEITH, ARDEAN, University of Rochester 

LIPSON, ROBERT A., Columbia University 

LISMAN, JOHN Massachusetts Institute of Technology 

LOVEDAY, KENNETH S., Massachusetts Institute of Technology 

LUZZATI, ANNE, Orsay, France 

MAcKAY, ALEXANDER R., Princeton University 

MAZAL, DENNIS, Rutgers University 

McCALL, PETER, Yale University 

MILCH, JAMES ROGER, Princeton University 

MINECONZO, GARY A., Union College 

MIYAMOTO, DAVID MARK, Duke University 

MOBBERLY, DEBORAH KAY, Tulane University 

MOORE, MARILYN R., University of Connecticut Health Center 

MOOSEKER, MARK S., University of Pennsylvania 

MORAN, NAVA, The Hebrew University 

NATALINI, JOHN J., Northwestern University 

NOONAN, KENNETH D., Princeton University 

O'RAND, ANGELA M., Temple University 

O'RAND, MICHAEL G., Temple University 

ORSINI, ROGER A., University of Connecticut 

OSMAN, RICHARD W., University of Chicago 

PARMENTIER, JAMES L., University of California, Santa Barbara 

PATTON, ALICE, University of Texas at Austin 

PENCEK, TERRENCE L., Illinois Institute of Technology 

PERRELET, ALAIN, The Rockefeller University 

PERSELL, ROGER, Hunter College 

PILAPIL, C., University of Montreal 

PREDDIE, ENRIQUE C., University of Montreal 

REUSCH, VICTOR M., Princeton University 

ROBERTS, JAMES T., Massachusetts General Hospital 

ROBERTSON, MRS. C. W., American Museum of Natural History 

ROSEN, JEFFREY B., Case Western Reserve University 

ROWLAND, ANDY, University of Pennsylvania 

SACCONE, JAMES M., University of Connecticut Medical School 

SAPIN, PAUL, Cleveland, Ohio 

SCHNUR, THOMAS, Oakland University 

SCOTTO, JOSEPH M., Institute for Basic Research, Staten Island 

SHAPIRO, EDWARD J., State University of New York at Buffalo 

SHAPIRO, ROBERT ALAN, Oakland University 

SHERMOEN, ANTONY W., Wesleyan University 

SHICK, J. MALCOLM, College of William and Mary 

SHIROKY, DOROTHY V., Johns Hopkins University 

SNYDER, DAVID A., Brown University and University of Southern California Medical School 

SNYDER, THOMAS P., Juniata College 



REPORT OF THE DIRECTOR 43 

SPERLING, LINDA, Harvard University 

STERNFELD, JOHN, University of California, San Diego 

STEVENS, E. D., University of Hawaii 

STILLER, RONALD A., Boston University 

STILLINGS, SUSAN N., Oberlin College 

STILLINGS, WAYNE, Oberlin College 

STRICKHOLM, STEVE, Indiana University 

SULANOWSKI, JACEK, Wayne State University 

SUSSMAN, PAUL, Columbia University 

SWANSON, RUTH ANN, Wayne State University 

SZAMIER, R. BRUCE, Albert Einstein College of Medicine 

SZONYI, ESZTER I., Boston University School of Medicine 

SZUMILAS, DIANA, Wayne State University 

TAYLOR. GRANTLEY W., New York University School of Medicine 

TSANG, MONICA LIK-SHING, Brandeis University 

TSIRINGAS, HARISSIOS F., State University of New York at Buffalo 

TUCKER, GAIL SUSAN, University of Kansas 

TURANSKY, DAVID G., Columbia University 

TURETSKY, OXANA, Brooklyn College 

UPPAL, J. S., University of Montreal 

VAN HOLDE, MARY ANNETTE, University of Oregon 

WAGNER, GEORGE J., State University of New York at Buffalo 

WECK, STEVEN D., New York University Medical School 

WEISS, JONATHAN, Wesleyan University 

WETHERBEE, RICHARD, University of Michigan 

WEXLER, ANDREW, Dartmouth College 

WINTEMUTE, GAREN JOHN, Yale University 

WOODARD, BRETT H., University of Pittsburgh 

YOUNGDAHL, PAMELA E., University of Oregon 

YULO, TERESA S., University of Rochester 

ZAKEVICIUS, JANE M., New York University Medical Center 

ZDUNSKI, H. DULEINE, Case Western Reserve University 

Library Readers, 1971 

ALLEN, GARLAND E., Assistant Professor of Biology, Washington University 
ALLEN, ROBERT DAY, Professor and Chairman, Department of Biological Sciences, State Uni- 
versity of New York at Albany 

ANDERSON, RUBERT S., Independent Library Reader, Marine Biological Laboratory 
BALL, ERIC G. Professor Emeritus of Biological Chemistry, Harvard University 
BENDET, IRWIN J., Professor of Biophysics, University of Pittsburgh 
BERLIN, RICHARD D., Associate Professor of Physiology, Harvard Medical School 
BERNE, ROBERT M., Professor and Chairman, Department of Physiology, University of Virginia 

School of Medicine 

BOETTIGER, EDWARD G., Professor of Physiology, University of Connecticut 
BRIDGMAN, ANNA JOSEPHINE, Professor and Chairman, Department of Biology, Agnes Scott 

College 

BUCK, JOHN, Chief, Laboratory of Physical Biology, National Institutes of Health 
CARLSON, FRANCIS D., Professor of Biophysics, Johns Hopkins University 
CASSIDY, FR. JOSEPH D., Assistant Professor of Biology, University of Notre Dame 
CHILD, FRANK M., Associate Professor of Biology, Trinity College 
CLARK, ARNOLD M., Professor of Biological Sciences, University of Delaware 
COHEN, SEYMOUR S., Professor and Chairman, Department of Therapeutic Research, University 

of Pennsylvania, School of Medicine 

COPELAND, DONALD EUGENE, Professor of Biology, Tulane University 
COUCH, ERNEST F., Assistant Professor of Biology, Texas Christian University 
CROWELL, SEARS, Professor, Department of Zoology, Indiana University 
DAVIS, BERNARD D., Professor of Bacterial Physiology, Harvard Medical School 
DUDLEY, PATRICIA L., Associate Professor of Biology, Barnard College 



44 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

EBNER, FORD F., Associate Professor, Brown rni\iTMt\ 

EDER, HOWARD A., Professor of Medicine, Albert Einstein College of Medicine 

EISEN, HERMAN N., Professor of Microbiology, \\ '.Islington University 

GABRIEL, MORDECAI L., Professor and Chairman, Biology Department, Brooklyn College 

GELFANT, GRACIELA C. CANDELAS, Professor, Department of Biology, University of Puerto Rico 

at Rio Piedros 
GERMAN, JAMES L., Investigator, and Director of the Laboratory of Human Genetics, The New 

York Blood Center and Associate Professor, Cornell University Medical College 
GINSBERG, HAROLD S., Professor and Chairman, Department of Microbiology, University of 

Pennsylvania 

GREEN, JAMES \V., Professor of Physiology, Rutgers University 
GROSS, PAUL R., Professor of Biology, Massachusetts Institute of Technology 
GUSSIN, ARNOLD E. S., Assistant Professor, Smith College 

HASTINGS, J. Woodland, Professor, Biological Laboratories, Harvard University 
HILLMAN, NINA \\ ., Research Associate, Temple University 
HILLMAN, RALPH, Professor, Temple University 
HOLTZER, HOWARD, Professor, University of Pennsylvania 
JOHNSON, EDWARD A., Graduate Student, University of New Hampshire 
KEMPTON, RUDOLF T., Professor Emeritus of Biology, Vassar College 

KEOSIAN, JOHN, Professor Emeritus, Rutgers University, The State University of New Jersey 
KIRSCHENBAUM, DONALD M., Associate Professor of Biochemistry, College of Medicine Downstate 

Medical Center 
KRASSNER, STUART M., Vice Chairman Department of Developmental and Cell Biology and 

Associate Professor, University of California, Irvine 

KRAVIT/, EDWARD A., Professor of Neurobiology, Harvard Medical School 
LICE, HAROLD H., Assistant Professor, University of Toledo 
LEVY, ARTHUR L., Chief of Clinical Chemistry, St. Vincent's Hospital and Medical Center of New 

York 

LURIA, SALVADOR E., Institute Professor of Biology, Massachusetts Institute of Technology 
MAKSLAND, DOUGLAS, Research Professor Emeritus, New York University 
MAUTM-.K, HENRY G., Chairman, Department of Biochemistry and Pharmacology, Tufts I'ni- 

versity School of Medicine 

MIZELL, MERLE, Professor of Biology, Tulane University 

MORRELL, FRANK, Professor of Neurology and Psychiatry, New York Medical College 
NASATIR, MAIMON, Professor of Biology, Lhiiversity of Toledo 

PERSON, PHILIP, Chief, Special Research Laboratory, Veterans Administration Hospital, Brooklyn 
PIKE, EILEEN H., Associate Professor of Parasitology, New York Medical College 
PLOUGH, HAROLD H., Professor of Biology Emeritus, Amherst College 
ROTH, JAY S., Professor of Biochemistry, University of Connecticut 
ROTH, L. EVANS, Professor and Director, Division of Biology, Kansas State University 
ROTH, OWEN H., Head, Department of Biology, St. Vincent College 
ROWLAND, LEWIS P., Professor and Chairman, Department of Neurology, University of 

Pennsylvania 

RUBINOW, S. L, Professor of Biomathematics, Cornell University Medical College 
RYBICKA, KRYSTYNA, Research Associate, Rice University 
ScHLESlNGER, R. WALTER, Professor of Chemistry, Rutgers Medical Center 
SCOTT, ALAN, Professor of Biology, Colby College 
SHEMIN, DAVID, Professor of Biochemistry, Northwestern University 
SILUNAS, REST AS E., Associate Professor of Sociology, Pennsylvania State University 
SMELSER, GEORGE K., Professor of Anatomy, Columbia University College of Physicians and 

Surgeons 

SONNENBLICK, B. P., Professor of Zoology, Rutgers University 

SONNENBLICK, EDMUND H., Associate Professor of Medicine, Harvard Medical School 
STETTEN, DE\\ ITT, JR., Director, NIGMS, National Institutes of Health 
STETTEN, MARJORIE R., Chemist, NIAMD, National Institutes of Health 
SIKAUSS, ELLIOTT W., Associate Professor of Medical Sciences, Brown University 
STRITTMATTER, PHILIPP, Professor of Biochemistry, University of Connecticut Health Center 
TEREBEY, NICHOLAS, Lecturer, Department of Biological Structure, University of Washington 



REPORT OF THE DIRECTOR 45 

WAINIO, WALTER, Professor and Chairman, Department of Biochemistry, Rutgers-The State 

University of New Jersey 

WEISS, LEON, Professor of Anatomy, Johns Hopkins Medical School 
WHEELER, GEORGE E,, Associate Professor of Biology, Brooklyn College 
WICHTERMAN, RALPH, Professor of Biology, Temple University 
WILSON, THOMAS HASTINGS, Professor of Physiology, Harvard Medical School 
WITTENBERG, JONATHAN B., Professor of Physiology, Albert Einstein College of Medicine 
YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate Medical 

Center 
ZEIDENBERG, PHILLIP, Instructor in Psychiatry, Columbia University, College of Physicians and 

Surgeons 

Students, 1971 

All students listed completed the formal course program. Asterisk indicates students com- 
pleting [lost-course research program. 

ECOLOGY 

AVISE, JOHN C., University of Texas, Austin 
*BALDWIN, RALPH W., Clark University 
*CANNON, JOHN C., Williams College 

CONNELL, MARY U., Kent State University 
*EARLANDSON, RALPH P., University of Chicago 

EIRIKSDOTTIR, GuoNY, Vassar College 

GAVIS, JEROME, Johns Hopkins University 
*JACOBS, NORMAN M., University of Pennsylvania 

MESCHER, MATTHEW F., Harvard LTniversity 
*MUNDY, PHILLIP R., University of Alabama 
*MOUSALLI, ELIE, American University, Beirut 

SHULENBERGER, ERIC, University of Kansas 
*SZEKELY, DANIEL R., State University of New York at Stony Brook 

THORSON, STUART H., University of Washington 
*\VAITE, THOMAS D., Harvard University 

*\VIDEGREN, ELISABETH J., Mississippi State College for Women 
*WiLSON, DAVID S., University of Rochester 
* \\OKLEY, ANN C., Yale LTniversity 

EMBRYOLOGY 

*ALBERTINI, DAVID F., University of Massachusetts, Amherst 
*BEEBE, DAVID C., University of Virginia 

BENECCHI, JOHN L., Marquette University 

*BRANDRIFF, BRIGITTE F., University of California, Santa Crux 
*CALVET, JAMES P., University of Connecticut 
*CLARK, STEPHEN H., Wesleyan LIniversity 
*COLBERT, DONALD A., Brown University 
*EVANS, LEONARD E., Michigan State University 
*GALLER, LYNNE, University of Chicago 
*HEUSER, MARSHA, Georgetown University 
*JOHNSON, LINDA M., Harvard University 
*KERN, CLIFFORD H., Ill, Indiana University 

KNAACK, LORETTA J., Rice University 
*KORENBERG, JULIE R., University of Wisconsin 
*KORNFELD, STEPHEN J., L^nion College 

LEE, FRANK G., Oberlin College 
*LEWIN, DAVID L, Yale University 

MACMORRIS, MARGARET A., California Institute of Technology 

MILLER, ELIZABETH T., University of Illinois 

NEWMAN, GERALDINE R. GIOSA, Medical College of Pennsylvania 
*PARENT, JAMES B., University of Virginia 



46 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

*ROSENBERG, PAUL A., Albert Einstein College of Medicine 
*SINGLEY, CART T., University of Hawaii 

SWEADNER, KATHLEEN, University of California, Santa Barbara 
*TANAKA, KAREN J., University of Minnesota 

WARREN, AUDREY J., Princeton University 

WEINBAUM, GEORGE, Albert Einstein Medical Center 

WOODWARD, JOHN B., Ill, University of Colorado 

EXPERIMENTAL BOTANY 

*AGHAJANIAN, JOHN G., Long Island University 

*BECK, TIMOTHY A., University of California, Los Angeles 

BEDIGIAN, DOROTHEA, University of Vermont 

CHECKLEY, DAVID M., JR., Scripps Institution of Oceanography 
*CLARK, ROBERT L., University of Cincinnati 

COHEN, DAN, Brandeis University 

DAVIDSON, JEFFREY N., Indiana University 
*DERR, JANICE A., Oberlin College 
*DEVrns, JANET, Duke University 
*FETSCHER, CHARLES T., Hamilton College 
*FINEMAN, ELLIOTT L., Oberlin College 
*GAUSS, VERENA, Mt. Holyoke College 
*GOLDSTEIN, MARJORIE F., Rutgers University 
*KiNG, DAN O., JR., Indiana University 

KLEIN, NANCY C., University of Virginia 

KLEYN, JOHN G., University of Puget Sound 

McCoNNELL, MAUREEN, University of Windsor 

OGUS, JUDITH R., Bennington College 

PEAVEY, D WIGHT G., Brandeis University 

SIMON, JACK H., State University of New York at Stony Brook 

SINGER, ELLEN M., University of Massachusetts 

WALKER, FREDERICK J., University of California, San Diego 

PHYSIOLOGY 

*ABRAHAMS, SUSAN JANE, Columbia University 
*BiROC, SANDRA LYN, Johns Hopkins University 
*BLUM, DR. HAYWOOD, Drexel University 
*BUZASH, ELIZABETH A., University of Connecticut 
*CLAY, JOHN R., University of Rochester 

COLTON, CAROL A., Rutgers Medical School 
*CoOFER, JON C., University of Wisconsin 
*CURRENT, STEVEN P., University of Chicago 
*DOMANIK, RICHARD A., Northwestern University 
*EATON, BARBRA L., University of Pennsylvania 
*EVANS, FREDERICK E., State University of New York at Albany 
*GORDON, CHARLES R., Massachusetts Institute of Technology 
*GULATI, JAGDISH, Pennsylvania Hospital 

HUSZAR, GABOR D., M.D., Boston Biomedical Research Institute 

KRANTZ, ALLEN, State University of New York at Stony Brook 
*LINNEY, ELWOOD, University of California, San Diego 

McELROY, JAMES D., University of California, San Diego 
*MEYERS, JUDY A., University of Pittsburgh 
*MiLLER, MICHAKL R., Milton S. Hershey Medical Center 
*PASTUSZYN, ANDRZEJ, University of New Mexico 
*PEARSON, TERRY W., University of California, Davis 
*PoPOT, jEAN-Luc, Institut Pasteur, Paris 



REPORT OF THE DIRECTOR 

REICHERT, THOMAS A., Carnegie- Mellon University 
*REID, LOLA C. McAoAMS, University of North Carolina, Chapel Hill 
*SACEVICH, EUGENE G., Johns Hopkins University 
*SALMON, EDWARD D., University of Pennsylvania 

*SIEGAL, MICHAEL S., Columbia University, College of Physicians and Surgeons 
*SIMPSON, PETER A., Brandeis University 
THOMAS, JOSEPH M., JR., Michigan State University 
*VAN SAMBEEK, JEROME W., Washington University, St. Louis 
*WALLACE, DOUGLAS C., Yale University 
*WARNER, CYNTHIA K., University of Tennessee 

WELCH, GEORGE R., University of Tennessee 

INVERTEBRATE ZOOLOGY 

*ANG, ESTRELLA Z., University of Pittsburgh 

ATKINSON, JOHN M., University of Hawaii 

BATES, ROBERT J., Yale University 

CLARK, ALVIN J., University of California, Berkeley 
*CORNELL, JOHN C., University of California, Berkeley 

COUTCHIE, PAMELA A., University of California, Davis 

DIEFENBACH, CARLOS O. DA C., State University of New York at Buffalo 
*DOUGHERTY, JAMES J., Ill, Rice University 

DOHRMANN, JOHN D., National Marine Fisheries Service Groundfish Biology 

DUDEK, FRANCIS E., University of California, Irvine 
*ELLISON, ANTHONY M., Yale University 

EVANS, STEPHEN J., University of California, Riverside 

EYMAN, KAREN A., Kent State University 
*FENNER, DOUGLAS H., Reed College 

HILL, MARGARET C., University of Kansas 

HUFF, ANNIE L., Fort Valley State College 
*!TAYA, STEPHEN K., University of Tennessee 

JOHNSON, GARY L., San Fernando Valley State College 
*KNUDSEN, ERIC L, University of California, Santa Barbara 

LEFLORE, WILLIAM, Atlanta University 

MARZOUK, JOSEPH B., Princeton University 

MILLER, JAMES R., Pennsylvania State LTniversity 

OWEN, PAUL H., JR., State University of New York at Syracuse 
*PALMER, LUCY B., Smith College 

SAMMARCO, PAUL W., Syracuse University 
*SHAPIRO, ELI, Yale University 
*TORRES, JOSEPH J., College of William and Mary 

WEN, GEORGE WALTER SUN, Harvard University 

WESTROM, WENDY K., Douglass College 

WILSON, MAXINE L., Emory University 
*WINQUIST, RAYMOND J., University of California, Santa Barbara 

NEUROBIOLOGY 

CLUSIN, WILLIAM T., Albert Einstein College of Medicine 

HOCHSTEIN, SHAUL STEPHEN, University of Jerusalem, Israel 

JOYNER, RONALD W., Duke University 

MORAN, DAVID T., University of Colorado 

RUSSELL, JOHN M., University of Utah 

SANES, JOSHUA R., Harvard University 

TWEEDLE, CHARLES D., Yale University 

ZARET, WENDY N., New York University Medical School 



48 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

FRONTIERS IN RESEARCH AND TEACHING 

ANEKWE, GREGORY, Tuskegee Institute 
FERGUSON, THOMAS, Delaware State College 
HUFF, ANNIE L., Fort Valley State College 
LEFLORE, WILLIAM B., Spelman College 
RACE, JAMES, JR., Texas Southern University 
TOUNSEL, JAMES G., Virginia State College 
WALKER, CHARLES A., Tuskegee Institute 



4. FELLOWSHIPS AND SCHOLARSHIPS, 1971 

The Crocker Scholarship: 

KLIE MOUSALLI, Ecology Course 
VERENA GAUSS, Botany Course 

The Jacobs Scholarship: 

JEAN-LUC POPOT, Physiology Course 
EUGENE G. SACEVICH, Physiology Course 

The Memhard Scholarship: 

DONALD A. COLBERT, Embryology Course 



5. TRAINING PROGRAMS 

FERTILIZATION AND GAMETE PHYSIOLOGY RESEARCH TRAINING PROGRAM 

I. INSTRUCTORS 

CHARLES B. METZ, University of Miami, Program Chairman 

MARCO CRIPPA, Laboratorio di Embriologia Molecolare, Naples, Italy 

GERTRUDE W. HINSCH, University of Miami 

LAJOS PIKO, Veterans Administration Hospital, Sepulveda, California 

ALLEN W. SCHUETZ, Johns Hopkins University 

WILLIAM H. TELFER, University of Pennsylvania 

II. LABORATORY ASSISTANTS 

MRS. CAROLYN CON WAY, Electron Microscope Assistant 
MR. STEVE SENFT, Photographic Assistant 
MRS. ANGELA O'RAND, Program Secretary 

III. TRAINEES 

ARAKELIAN, HELEN, Michigan State University 

BANTLE, JOHN A., Ohio State University 

BERGSTROM, BEVERLY H., University of North Carolina 

BISCHOFF, WILLIAM L., University of North Carolina 

CAYER, MARILYN L., University of Miami 

CLEGG, KERRY B., University of California, Los Angeles 

CONWAY, ARTHUR F., University of Miami 

COOPER, ALAN DOUGLAS, Worcester State College 

ECKLUND, PETER S., Wayne State University 

GOULD, STANLEY F., Wayne State University 

HOWE, CRAK., King's College, University of Cambridge, England 



REPORT OF THE DIRECTOR 49 

KAHN, JAMES L., University of Toledo 

LAMARCA, MICHAEL J., Lawrence University 

MANN, WILLIAM J., JR., Pennsylvania State College of Medicine 

MATSUMOTO, LLOYD H., St. Louis University 

SHIPPEE, ELIZABETH, Cornell University 

WASSARMAN, PAUL M., Purdue University 

IV. LECTURES 

W. H. TELFER Function of nurse cells and follicular epithelium in insect ovaries 

G. W. DUNCAN The effects of prostaglandin on reproductive functions 

M. CRIPPA Mechanism for ribosomal gene amplification 

F. H. BRONSON Pheromones and mammalian reproduction 

B. G. BRACKETT In vitro fertilization of mamalian ova 

K. A. LAWRENCE Antibodies to gonadotropins and their effect on gonadotropin action and 

fertility 

D. W. FAWCETT The organization of the mammalian seminiferous epithelium 
I. DAWID The function of mitochondrial DNA in frog eggs and embryos 

D. N. WARD The ammo acid sequence of ovine and bovine luteinizing hormone 

V. SPECIAL LECTURES 

MURIEL H. W T ALKER The arrangement of nucleoprotein in elongate sperm heads 
JOHN BIGGERS Metabolic changes in early mammalian development 

YOSEF ALONI Transcription of mitochondrial DNA in HeLa cells 

EXCITABLE MEMBRANE PHYSIOLOGY AND BIOPHYSICS TRAINING PROGRAM 

I. INSTRUCTORS 

W. J. ADELMAN, JR., Director, Professor of Physiology, University of Maryland School of Medicine 

C. ARMSTRONG, Professor, University of Rochester School of Medicine 

J. W. MOORE, Professor of Physiology, Duke University School of Medicine 

T. NARAHASHI, Professor of Physiology, Duke University School of Medicine 

Y. PALTI, Associate Professor of Physiology, The Technion Medical School, Haifa, 

II. CONSULTANTS 

K. S. COLE, Senior Research Biophysicist, MINDS, National Institutes of Health 
L. J. MULLINS, Professor of Biophysics, University of Maryland School of Medicine 

III. TRAINEES 

BORG, DR. SIDNEY F., Stevens Institute of Technology 

BRUTSAERT, DR. DIRK L., University of Antwerp 

FAN, DR. CHUNGPENG, Rutgers University 

FROELICH, MR. OTTO, University of Toledo 

GARDNER, DR. COLIN R., Harvard Medical School 

GOLDFINGER, MR. MsLViN D., University of Maryland Medical School 

GRIFFIN, PATRICIA M., University of Pennsylvania 

HARBUS, MR. FREDERICK L, Massachusetts Institute of Technology 

MICHAELS, MR. DAVID W., University of Delaware 

ROSE, DR. MARY C., Duke University 

SIDIE, DR. JAMES M., JR., Indiana University 

STARXAK, DR. MICHAEL E., State University of New York at Binghamron 



50 



\\NUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



IV. LECTURES 
T. SCHWARTZ 



N. C. HEBERT 
S. WATSON 
K. S. COLE 

J. W. MOORE 



Y. PALTI 



W. K. CHANDLER 



L. COHEN 

C. ARMSTRONG 



G. EHRENSTEIN 

1. TASAKI 

W. STOECKENIUS 



H. LECAR 

L. MULLINS 



D. GlLHI K I 

W. ADELMAN 

F. A. DODGE 
T. NARAHASHI 



H. GRUNDFEST 
A. B. STEINBACH 



The flux equation its rationale and relation to diffusion regimes 

The Ussing-Teorell "Unidirectional Flux Equation" 

The Gibbs-Donnan equilibrium 

The Goldman equation with and without active transport and/or constant 

field conditions 

Simple diffusion regimes relation to electric equivalent circuits 
Properties of ion sensing glass micro-electrodes 
Evolution of structure in membranes of nitrifying bacteria 
Biophysics and a nerve impulse 
Voltage clamp strategy 
Voltage clamp tactics 
Voltage clamp arrangements 
Ionic current kinetics 
The Hodgkin-Huxley axon 

Action potential reconstruction and propagation 
Varying potential voltage clamps 
General properties of internally perfused atoms 
Perfusion of axons with solutions of low ionic strength 
Ionic selectivity of the axonal membrane 
Properties of the delayed rectifier 

Sodium conductance with K-free fluoride solutions inside the axon 
Birefringence and fluorescence changes during axon activity 
Light scattering changes in axons 
The K channels of nerve and other excitable tissues 
Interactions of TEA and TEA derivatives with the K channels, and what this 

tells about the nature of the channels I. 
Interactions of TEA and TEA derivatives with the K channels, and what this 

tells about the nature of the channels II. 
Artificial membranes. I. Structure 
Artificial membranes. II. Specificity 
Artificial membranes. III. Excitability 
Macromolecular approach to nerve excitation 
Fluorescence studies of nerve excitation 
Membrane structure I. 
Membrane structure II. 
Membrane structure discussion 
Noise and fluctuations in nerve membranes 
Recovery processes in axons-active ion transport 
The interaction between diffusion and chemical forces in determining the 

resting membrane potential 

Inhibitors of Na + and K + currents-inferences regarding mechanisms 
Models for nerve excitation 
Membrane surface charges I. 
Membrane surface charges II. 

Properties of the periaxonal space in modifying membrane behavior 
Properties of the perineuronal space in modifying neuronal behavior 
Receptor potentials 
Mode of action of drugs on excitable membranes. I. General consideration 

and tetrodotoxin 
Mode of action of drugs on excitable membranes. II. Toxins, anesthetics, 

insecticides and enzymes 

Characteristics of end-plate membrane conductances 
Electrically excitable membranes 
Electrically inexcitable membranes 

Neuromuscular transmission. I. Presynaptic transmitter release 
Neuromuscular transmission. II. Postsynaptic response 



REPORT OF THE DIRECTOR 



51 



6. TABULAR VIEW OF ATTENDANCE, 1967-1971 

1967 1068 1969 



INVESTIGATORS TOTAL ......... ................ 590 

Independent ................. 313 

Library Reader. ... 78 

Research Assistants. ............ 



STUDENTS TOTAL .............. ............. 132 

Invertebrate Zoology ......... 41 

Embryology ..................................... 20 

Physiology ...................................... 31 

Experimental Botany ............................. 20 

Ecology ........................................ 20 



TRAINEES TOTAL . 



16 



TOTAL ATTENDANCE 738 

Less Persons represented in two categories 4 



734 

INSTITUTIONS REPRESENTED TOTAL 177 

FOREIGN INSTITUTIONS REPRESENTED. 29 



528 

281 

76 

171 

122 
39 
20 
30 
15 
18 

17 

667 

7 

660 
169 

23 



566 

310 

68 

188 

118 
35 
20 
30 
16 
17 

29 

713 

5 

708 

187 

24 



1970 1971 



532 

324 

73 

135 

142 
41 
28 
31 
19 
23 

33 

707 


707 

191 

21 



554 

322 

76 

156 

130 

29 
28 
33 

22 
18 

44 

728 


728 
219 

27 



7. INSTITUTIONS REPRESENTED, 1971 



Agnes Scott College 

Albert Einstein College of Medicine 

American Museum of Natural History 

Amherst College 

Barnard College 

Bennington College 

Boston City Hospital 

Boston College 

Boston University 

Boston University School of Medicine 

Brandeis University 

Brooke Army Medical Center 

Brooklyn College, The City University of New 

York 

Brown University 
Bucknell University 
California, University of, Berkeley 
California, University of, Davis 
California, University of, Irvine 
California, University of, Los Angeles 
California, University of, Riverside 
California, University of, San Diego 
California, University of, Santa Barbara 
California, University of, Santa Cru/ 
California Institute of Technology 
Cambridge, University of 
Carnegie Institution of Washington 
Carnegie-Mellon University 
Case Western Reserve University 
Chicago, University of 
Cincinnati, University of 



City College, The City University of New 

York 

Clark University 
Colby College 

College of William and Mary 
Colorado, University of 
Colorado, University of, Medical Center 
Columbia University 
Columbia University, College of Physicians and 

Surgeons 

Connecticut, University of 
Connecticut, University of, Health Center 
Connecticut, University of, Medical School 
Cornell University 
Cornell University Medical College 
Dartmouth College 
Dartmouth Medical School 
Davidson College 
Delaware, University of 
Douglass College 
Drew University 
Drexel University 
Duke University 
Duke LJniversity Medical Center 
East Stroudsburg State College 
Emory University 
Florida, University of 
Florida Atlantic University 
Florida State University 
Goucher College 
Georgetown University 



52 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



I lamilton College 

Harvard Medical School 

Harvard University 

Hawaii, University of 

Hunter College, The City University of Nr\\ 

York 

Idaho, University of 
Illinois, University of 
Illinois Institute of Technology 
Immaculata College 
Indiana University 
Institute for Basic Research in Mental 

Retardation 

Institute for Cancer Research, The 
Institute for Muscle Research, Inc. 
Iowa, University of 
Iowa State University 
Johns Hopkins University, The 
Johns Hopkins University, The, School of 

Medicine 
Juniata College 
Kansas, University of 
Kansas State LIniversit) 
Kent State Universiix 
Kentucky, University ot 
Keuka College 
Lawrence University 
Lehman College, The City University of Ne\\ 

York 

Long Island University 
Louisiana State University 
Maine, University of 
Marquette University 
Marine Research Foundation, Inc. 
Maryland, University of 

Maryland, University of, School of Medicine 
Massachusetts, University of 
Massachusetts General Hospital 
Massachusetts Institute of Technology 
Medical College of Ohio at Toledo 
Medical College of Pennsylvania 
Mellon Institute of the Carnegie-Mellon 

University 

Miami, University of 
Miami University 
Michigan, University of 
Michigan State University 
Millersville State College 
Milton S. Hershey Medical Center 
Minnesota, University of 
Mississippi State College for Women 
Mount Holyoke College 
Mount Sinai School of Medicine, The City 

University of New York 
National Institute of Mental Health 
National Institutes of Health 
National Marine Fisheries Service 



\ew Hampshire, University of 
New Mexico, University of 
New Mexico, University of, School of Medicine 
New York Blood Center, The 
New York Medical College 
New York University College of Dentistry 
New York University Medical College 
North Carolina, University of 
North Carolina State University of Raleigh 
Northwestern University 
Notre Dame, L T niversity of 
Oak Ridge National Laboratory 
Oakland University 
Oberlin College 
Ohio State University 
Ohio University 
Oregon, University of 
Pennsylvania, Ihiiversity of 
Pennsylvania, University of, School of Medicine 
Pennsylvania Hospital 
Pennsylvania State University 
Pittsburgh, University of 
Pomona College 
Princeton University 
Puget Sound, University of 
Purdue University 

Queens College, The City University of New 
" York 
Reed College 

Rhode Island, University of 
Rice University 
Rochester, University of 
Rochester, University of, Medical School 
Rockefeller University, The 
Rutgers Universit \ 
Rutgers University Medical School 
St. Elizabeth Hospital 
St. Louis University 
St. Vincent College 
St. Vincent's Hospital and Medical Center of 

New York 

San Fernando Valley State College 
Scripps Institution of Oceanography 
Smith College 

South Florida, University of 
Southern California, L T niversity of, Medical 

School 

Stanford University 
St.ite LTniversity of New York, Downstate 

Medical Center 
State University of New York, Upstate Medical 

Center 

State University of New York at Albany 
State University of New York at Buffalo 
State University of New York at Stony Brook 
Syracuse University 
Temple University 



REPORT OF THE DIRECTOR 



53 



Tennessee, University of 

Texas, University of 

Texas, University of, at Austin 

Texas Christian University 

Toledo, University of 

Trinity College 

Tufts University 

Tulane University 

Union College 

Upsala College 

Utah, University of 

Vassar College 

Vermont, University of 

Veterans Administration Hospital, Brooklyn 

Veterans Administration Hospital, California 

Virginia, University of 

Virginia, University of, School of Medicine 

Wake Forest University 

Washington, University of 

Washington University 

Washington University School of Medicine 

Wayne State University 

Wesleyan University 

Williams College 

Wisconsin, University of 

Wistar Institute 

Woods Hole Oceanographic Institution 

Worcester State College 

Yale University 

Yale University School of Medicine 



FOREIGN INSTITUTIONS REPRESENTED, 1971 

American University of Beirut, Lebanon 

Antwerp, University of, Belgium 

Calgary, University of, Canada 

Cambridge, University of, England 

CXR Laboratory of Molecular Embryology, 

I tab- 
Hebrew University, The, Israel 
Hebrew University Medical School, Israel 
Jerusalem, University of, Israel 
Leeds, University of, England 
London, University of, England 
McGill University, Canada 
Medical Research Council, England 
Moleculaire Institut Pasteur, France 
Montreal, University of, Canada 
National Institute for Medical Research, 

England 

Norwegian Institute of Technology, Norway 
Oslo, University of, Norway 
Ottawa, LIniversity of, Canada 
Oxford, University of, England 
Paris, University of, France 
Philips Research Laboratory, Holland 
Puerto Rico, University of, at Rio Piedies 
Queen Elizabeth College, University of London, 

England 

Royal College of Surgeons of England 
Toronto, University of Canada 
Trondheim, University of, Norway 
Windsor, University of, Canada 



8. FRIDAY KYKNINO LKCTUKKS, 1 ( )71 

July 2 

PRESTOX CLOUD.. . Biospheric, atmospheric and crustal evolution of 

University of California the primitive earth 

Santa Barbara 

July 8 

AHAROX KATCHALSKV. . Biothermodynamics and network analysis part I 

Weizmann Institute of Science 
Alexander Forbes Lecturer at MBL 



July 9 

AHARON- KATCHALSKY 



Biothermoclvnamics and network analysis part II 



July 16 

R. S. WOLFE. . 
University of Illinois 



Methyl transfer reactions in methane bacteria 
and their ecological significance 



54 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

July 23 

HARRY EAGLE pH, contact inhibition and cell metabolism 

Albert Einstein College of Medicine 
W. J. V. Osterhuut Memorial 
Lecture 

July 30 

MELVIN J. COHEN. . ..Some functional implications of neuronal ge- 

Yale University ometr\ 

August 6 

O. L. MILLER, JR.. Visualization of genes in action 

Oak Ridge National Laboratory- 
August 13 

SEYMOUR BEN/.ER. . . . . .Genes and the nervous system of Drosopldhi 

California Institute of Technology- 
August 20 

R. K. SELANDER Biochemical polymorphism and systematics 

University of Texas 
Rand Fellow at the MBL 

August 27 

MARTIN RAFF An immunological approach to lymphocytes and 

National Institute for Medical Re- the cell surface 

search, London 

Lillie Fellow at the MML 

0. TUESDAY EVENING SEMINARS, 1071 
August 10 

CHARLES B. METZ Mammalian sperm hyaluronidase, an isoantigen 

ALBERTO SEIGUER of possible interest for fertility control 

AMALIA CASTRO 

GERTRUDE W. HINSCH . Spermatocyte formation in the vas deferens of 

MURIEL H. \\*ALKER spider crabs 

MURIEL H. WALKER. ... . Microsporidia in the reproductive tract of Libia la 

GERTRUDE W. HINSCH dubia 

LEONARD NELSON Motility control mechanisms in Arbacia sperm 

August 17 

RICHARD L. MILLER Tricliopla.v adliacreus Schulze, 1883: return of an 

enigma 
SEYMOUR ZIGMAN. . . ... .Effects of near UY tryptophan photoproducts on 

proteins 

T. A. BORGESE . Iso-electric gel focusing of duck hemoglobins 

RICHARD EGNOR 

LASLO Z. BITO . . Concentrative accumulation of prostaglandins by 

DAVID TURANSKY some tissues of marine invertebrates and 

ALICE VAN VORIS vertebrates 



REPORT OF THE DIRECTOR 

10. MEMBERS OF THE CORPORATION, 1971 

Including Action of 1971 Annual Meeting 

Life Members 

ADOLPH, DR. EDWARD F., University of Rochester School of Medicine and Den- 
tistry, Rochester, New York 14620 

BEHRE, DR. ELINOR M., Black Mountain, North Carolina 28711 
BERTHOLF, DR. LLOYD M., 1228 Gettysburg Drive, Bloomington, Illinois 61701 
BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center, 

444 East 68 Street, New York, New York 10021 

BRADLEY, DR. HAROLD C, 2639 Durant Avenue, Berkeley, California 94704 
BRODY, MR. DONALD, 522 Fifth Avenue, New York, New York 10018 
COLE, DR. ELBERT C., 2 Chipman Park, Middlebury, Vermont 05753 
COWDRY, DR. E. V., 4580 Scott Avenue, St. Louis, Missouri 63110 
CRANE, MRS. W. MURRAY, 820 Fifth Avenue, New York, New York 10021 
DAWSON, DR. A. B., 12 Scott Street, Cambridge, Massachusetts 02138 
DAWSON, DR. J. A., 129 Violet Avenue, Floral Park, Long Island, New York 11001 
DILLER, DR. IRENE C., 2417 Fairhill Avenue, Glenside, Pennsylvania 19038 
DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania 19038 
HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, 

St. Louis, Missouri 63110 

HESS, DR. WALTER, 787 Maple Street, Spartanburg, South Carolina 29302 
HIBBARD, DR. HOPE, 366 Reamer Place, Oberlin, Ohio 44074 
HISAW, DR. F. L., 5925 S. W. Plymouth Drive, Corvallis, Oregon 97330 
HOLLAENDER, DR. ALEXANDER, Biology Division, Oak Ridge National Labora- 
tory, Oak Ridge, Tennessee 37830 

IRVING, DR. LAURENCE, University of Alaska, College, Alaska 99701 
LOWTHER, DR. FLORENCE, Barnard College, New York, New York 10027 
MACDOUGALL, DR. MARY STUART, Mt. Yernon Apartments, 423 Clairmont 

Avenue, Decatur, Georgia 30030 

MALONE, DR. E. F., 6610 North llth Street, Philadelphia, Pennsylvania 19126 
MANWELL, DR. REGINALD D., Department of Biology, Syracuse University, 

Syracuse, New York 13210 

PAGE, DR. I. H., Cleveland Clinic, Euclid at E. 93rd Street, Cleveland, Ohio 44106 
PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana 47405 
PLOUGH, DR. H. H., 15 Middle Street, Rt. 1, Amhersl, Massachusetts 01002 
POLLISTER, DR. A. W., Department of Zoology, Columbia University, New York, 

New York 10027 

POND, SAMUEL E., 53 Alexander Street, Manchester, Connecticut 06040 
PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania 

19104 

SCHRADER, DR. SALLY, Duke University, Durham, North Carolina 27706 
SEVERINGHAUS, AURO E., 375 West 250th Street, New York, New York 10071 
SMITH, DR. DIETRICH C., 218 Oak Street, Catonsville, Maryland 12128 
STRAUS, DR., W. L., JR., Department of Anatomy, The Johns Hopkins University 
Medical School, Baltimore, Maryland_21205 



56 \\NUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

STUNKARD, DR. HORACE W., American Museum of Natural History, Central Park 

West at 79th Street, New York, New York 10024 
TAYLOR, DR. WM. RANDOLPH, Department of Botany, University of Michigan, 

Ann Arbor, Michigan 48104 

TURNER, DR. C. L., Northwestern University, Evanston, Illinois 60201 
WAITE, DR. F. G., 144 Locust Street, Dover, New Hampshire 03820 
WALLACE, DR. LOUISE B., 359 Lytton Avenue, Palo Alto, California 94301 
WARREN, DR. HERBERT S., % Leland C. Warren, 721 Conshohocken State Road, 

Penn Valley, Pennsylvania 19072 
WILLIER, DR. B. H., Department of Biology, The Johns Hopkins University, 

Baltimore, Maryland 2121S 
YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts 02357 

Regular Members 

ABBOTT, DR. BERNARD C., Department of Biological Sciences, University of 

Southern California, University Park, Los Angeles, California 90007 
ADELBERG, DR. EDWARD A.. Department of Microbiology, Yale University 

Medical School, \V\\ Haven, Connecticut 06510 
ADELMAN, DR. WM. J., JR., Building 36 Room 2A 31, National Institutes of 

Health, Bethesda, Maryland 20014 
ALLEN, DR. GARLAND E., Biology Department, Washington University, St. Louis, 

Missouri 63103 
ALLEN, DR. ROBERT D., Department of Biological Sciences, State University of 

New York at Albany, Albany, New York 12203 
ALSCHER, DR. RUTH, Department of Biology, Manhattanville College, Purchase, 

New York 10577 

AMATNIEK, MR. ERNEST, 154 Bay Road, Huntington, New York 11743 
AMBERSON, DR. WILLIAM R., Katy Hatch Road, Falmouth, Massachusetts 02540 
ANDERSON, DR. EVERETT, Department of Anatomy and Laboratory of Human 

Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115 
ANDERSON, DR. J. M., Division of Biological Sciences, Emerson Hall, Cornell 

University, Ithaca, New York 14850 

ANDERSON, DR. HUBERT S., Box 113, Woods Hole, Massachusetts 02543 
ARMSTRONG, DR. CLAY M., Department of Physiology, University of Rochester, 

Rochester, New York 14603 
ARMSTRONG, DR. PHILIP B., Department of Anatomy, State University of New 

York, College of Medicine, Syracuse, New York 13210 
ARNOLD, DR. JOHN MILLER, Pacific Biomedical Research Center, 2538 The Mall, 

University of Hawaii, Honolulu, Hawaii 96822 
ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory, 

Oak Ridge, Tennessee 37830 

ASHWORTH, DR. JOHN MICHAEL, Deparlment of Biochemistry, Leicester Uni- 
versity, Leicester, England, U. K. 
ATWOOD, DR. KIMBALL C., Department of Human Genetics and Development, 

Columbia Universiu, College of Physicians and Surgeons, New York, New 

York 10032 

MR, DR. WALTER, Department of Biology, Rensselaer Polytechnic Institute, 
Troy, New York 12181 



REPORT OF THE DIRECTOR 57 

AUSTIN, DR. COLIN RUSSELL, Physiological Laboratory, Cambridge University, 

Downing Street, Cambridge, England, U. K. 

AUSTIN, DR. MARY L., 506^ North Indiana Avenue, Bloomington, Indiana 47401 
BACON, MR. ROBERT, Church Street, Woods Hole, Massachusetts 02543 
BAKALAR, MR. DAVID, 35 Lapland Road, Chestnut Hill, Massachusetts 02167 
BALL, DR. ERIC G., Department of Biological Chemistry, Harvard Medical 

School, Boston, Massachusetts 02115 
BALLARD, DR. WILLIAM W., Department of Biological Sciences, Dartmouth 

College, Hanover, New Hampshire 03755 
BANG, DR. F. B., Department of Pathobiology, The Johns Hopkins University 

School of Hygiene, Baltimore, Maryland 21205 
BARD, DR. PHILLIP, Department of Physiology, The Johns Hopkins University 

Medical School, Baltimore, Maryland 21205 
BARTELL, DR. CLELMER K., Department of Biological Sciences, Louisiana State 

University of New Orleans, New Orleans, Louisiana 70113 

EARTH, DR. LESTER G., Marine Biological Laboratory, Woods Hole, Massa- 
chusetts 02543 
BARTH, DR. LUCENA, Marine Biological Laboratory, Woods Hole, Massachusetts 

02543 
BARTLETT, DR. JAMES H., Department of Physics, University of Alabama, P.O. 

Box 1921, University, Alabama 35486 

BAUER, DR. G. ERIC, Department of Anatomy, University of Minnesota, Minne- 
apolis, Minnesota 55414 
BAYLOR, DR. E. R., State University of New York at Stony Brook, Stony Brook, 

Long Island, New York 11790 
BAYLOR, DR. MARTHA B., State University of Xew York at Stony Brook, Stony 

Brook, Long Island, New York 11790 
BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa 

City, Iowa 52240 
BECK, DR. L. Y., Department of Pharmacology, Indiana University, School of 

Experimental Medicine, Bloomington, Indiana 47401 
BELAMARICH, DR. FRANK A., Department of Biology, Boston University, Boston, 

Massachusetts 02215 
BELL, DR. ALLEN, Department of Anatomy, University of Colorado, Medical 

Center, Denver, Colorado 80220 

BELL, DR. EUGENE, Department of Biology, Massachusetts Institute of Tech- 
nology, Cambridge, Massachusetts 02139 
BENNETT, DR. MICHAEL V. L., Department of Anatomy, Albert Einstein College 

of Medicine, Bronx, New York 10461 
BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet 

Briar, Virginia 24595 
BERG, DR. WILLIAM E., Department of Zoology, University of California, 

Berkeley, California 94720 
BERMAN, DR. MONKS, National Institutes of Health, Institute for Arthritis and 

Metabolic Diseases, Bethesda, Maryland 20014 
BERNE, DR. ROBERT M., University of Virginia School of Medicine, Charlottes- 

ville, Virginia 22903 
BERNHEIMER, DR. ALAN W., New York University College of Medicine, New 

York, New York 10016 



58 ANNUAL REPORT OF THK MARINE BIOLOGICAL LABORATORY 

BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne State University 

College of Medicine, Detroit, Michigan 48237 
BERSOHN, DR. RICHARD, Department of Chemistry, Columbia University, 959 

Havemeyer Hall, New York, New York 10027 
BIGGERS, DR. JOHN DENNIS, The Johns Hopkins University School of Hygiene 

and Public Health, Division of Population Dynamics, Baltimore, Maryland 

21205 
BISHOP, DR. DAVID W., Medical College of Ohio at Toledo, P.O. Box 6190, 

Toledo, Ohio 43614 

BLANCHARD, DR. K. C, The Johns Hopkins University Medical School, Balti- 
more, Maryland 21205 

BLOCH, DK. ROBERT, Adalbertstr. 70-8, Munich, Germany (13) 
BLUM, DR. HAROLD F., Department of Biological Sciences, State University of 

New York at Albany, Albany, New York 12203 
BODIAN, DR. DAVID, Department of Anatomy, The Johns Hopkins University, 

709 North Wolfe Street, Baltimore, Maryland 21205 
BOELL, DR. EDGAR J., Department of Biology, Kline Biology Tower, Yale 

University, New Haven, Connecticut 06520 
BOETTIGER, DR. EDWARD G., Department of Zoology, University of Connecticut, 

Storrs, Connecticut 06268 
BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin, 

Texas 78712 

BOOLOOTIAN, DR. RICHARD A., Box 24787, Los Angeles, California 90024 
BOREI, DR. HANS G., Leidy Laboratory, Department of Biology, University of 

Pennsylvania, Philadelphia, Pennsylvania 19104 
BORSELLINO, DR. ANTONIO, Institute di Fiscia, Viale Benedetto XV, 5 Genova, 

Italy 
BOWEN, DR. VAUGHN T., W r oods Hole Oceanographic Institution, Woods Hole, 

Massachusetts 02543 
BRANDT, DR. PHILIP WILLIAMS, Department of Anatomy, Columbia University, 

College of Physicians and Surgeons, New York, New York 10032 
BRIDGMAN, DR. ANNA J., Department of Biology, Agnes Scott College, Decatur, 

Georgia 30030 

BRINLEY, DR. F. J., JR., Department of Physiology, The Johns Hopkins Uni- 
versity Medical School, Baltimore, Maryland 21205 
BRONK, DR. DETLEV W., The Rockefeller University, 66th Street and York 

Avenue, New York, New York 10021 
BROOKS, DR. MATILDA M., Department of Physiology, University of California, 

Berkeley, California 94720 

BROWN, DR. DUGALD E. S., 38 Whitman Road, Woods Hole, Massachusetts 02543 
BROWN, DR. FRANK A., JR., Department of Biological Sciences, Northwestern 

University, Evanston, Illinois 60201 
BROWN, DR. JOEL E., Department of Anatomy, School of Medicine, Vanderbilt 

University, Nashville, Tennessee 37203 
BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of 

Health, Bethesda, Maryland 20014 

BULLOCK, DR. T. H., Department of Neuroscience, University of California, 
i Diego, La Jolla, California 92038 



REPORT OF THE DIRECTOR 59 

BURBANCK, DR. MADELINE PALMER, Box 15134, Emory University, Atlanta, 
Georgia 30322 

BURBANCK, DR. WILLIAM I)., Box 15134 Emory University, Atlanta, Georgia 
30322 

BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilming- 
ton, Delaware 19805 

BURGER, DR. MAX M., Department of Biology, Princeton University, Princeton, 
New Jersey 08549 

BURNETT, DR. ALLISON LEE, Department of Biology, Northwestern University, 
Evanston, Illinois 60201 

BUSSER, DR. JOHN H., American Institute of Biological Sciences, 3900 Wisconsin 
Avenue NW, Washington, D. C. 20016 

BUTLER, DR. E. G., Department of Biology, Princeton University, Princeton, 
New Jersey 08540 

CANTONI, DR. GIULLIO, National Institutes of Health, Department of Mental 
Health, Bethesda, Maryland 20014 

CARLSON, DR. FRANCIS D., Department of Biophysics, The Johns Hopkins 
University, Baltimore, Maryland 21218 

CARPENTER, DR. RUSSELL L., 60-H Street, Winchester, Massachusetts 01890 

CARRIKER, DR. MELBOURNE R., Director, Systematics-Ecology Program, Marine 
Biological Laboratory, Woods Hole, Massachusetts 02543 

CASE, DR. JAMES F., Department of Biology, University of California, Santa 
Barbara, California 93106 

CASSIDY, REV. JOSEPH D., O.P., Department of Biology, University of Notre 
Dame, Notre Dame, Indiana 46556 

CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York Avenue, 
New York, New York 10021 

CHAET, DR. ALFRED B., University of W T est Florida, Pensacola, Florida 32505 

CHAMBERS, EDWARD L., University of Miami School of Medicine, Miami, Florida 
33146 

CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton, 
New Jersey 08540 

CHAUNCEY, DR. HOWARD 11., Veterans Administration Central Office, Washing- 
ton, D. C. 20420 

CHENEY, DR. RALPH H., Honorary Research Associate, Brooklyn Botanic 
Gardens, 1000 Washington Avenue, Brooklyn, New York 11225 

CHILD, DR. FRANK M., Department of Biology, Trinity College, Hartford, Con- 
necticut 06106 

CLAFF, DR. C. LLOYD, 506 N. Warren, Brockton, Massachusetts 02403 

CLARK, DR. A. M., Department of Biological Sciences, University of Delaware, 
Newark, Delaware 19711 

CLARK, DR. ELOISE E., National Science Foundation, 1800 G. Street, Washington, 
D. C. 20550 

CLARK, DR. LEONARD B., 149 Sippewissett Road, Falmouth, Massachusetts 
02540 

CLARKE, DR. GEORGE L., Biological Laboratories, Harvard University, Cam- 
bridge, Massachusetts 02138 

CLAYTON, DR. RODERICK K., Section of Genetics, Development and Physiology, 
Cornell University, Ithaca, New York 14850 



60 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

CLEMENT, DR. A. C., Department of Biology, Emory University, Atlanta, 

Georgia 30322 

CLOWES, DR. GEORGE H. A., JR., Harvard Medical School, Boston, Massa- 
chusetts 02115 
COHEN, Dr. ADOLPH I., Department of Opthalmology, Washington University, 

School of Medicine, 4550 Scott, St. Louis, Missouri 67110 
COHEX, DR. LAWRENCE B., Department of Physiology, Yale University, New 

Haven, Connecticut 06510 

COHEN, DR. SEYMOUR S., 635 Ash Street, Denver, Colorado 80220 
COLE, DR. KENNETH S., Laboratory of Biophysics, NINDS, National Institutes 

Of Health, Bethesda, Maryland" 20014 
COLLIER, DR. JACK R., Department of Biology, Brooklyn College, Brooklyn, 

New York 11210 
COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, 

New York, 11367 
COLWIN, DR. LAURA H., Department of Biology, Queens College, Flushing, New 

York 11367 

COOPERSTEIN, DR. SHERWIN J., School of Dental Medicine, University of Con- 
necticut, Hartford, Connecticut 06105 
COPELAND, DR. I). EUGENE, Department of Biology, Tulane University, New 

Orleans, Louisiana 70118 
CORNELL, DR. XKAL W., Department of Chemistry, Pomona College, Claremont, 

California 91711 

CORNMAN, DR. IVOR, 10A Orchard Street, Woods Hole, Massachusetts 02543 
COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 27514 
COSTELLO DR. HELEN MILLER, Department of Zoology, University of North 

Carolina, Chapel Hill, North Carolina 27514 
COUSINEAU, DR. GILLES H., Department of Biology, Montreal University, P. O. 

Box 6128, Montreal, P. Q., Canada 

CRANE, MR. JOHN O., Woods Hole, Massachusetts 02543 
CRANE, DR. ROBERT K., Department of Physiology, Rutgers Medical School, 

New Brunswick, New Jersey 08903 
CRIPPA, DR. MARCO, CNR Laboratory of Molecular Embryology, Arco Felice, 

Naples, Italy 
CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire 

03755 

CROUSE, DR. HELEN Y., Institute for Molecular Biophysics, Florida State Uni- 
versity, Tallahassee, Florida 32306 
CRO\VELL, DR. SEARS, Department of Zoology, Indiana L T niversity, Bloomington, 

Indiana 47401 
CSAPO, DR. ARPAD I., Washington University School of Medicine, 4911 Barnes 

Hospital Plaza, St. Louis, Missouri 63110 
DAIGNAULT, MR. ALEXANDER T., W. R. Grace and Company, 7 Hanover Square, 

New York, New York 10005 
DAN, DR. JEAN CLARK, I )epartment of Biology, ( )chanomizu University, Otsuka, 

Bunkyo-Ku, Tokyo, Japan 



KKI'ORT OF THE DIRECTOR 61 

DAN, DR. KATSUMA, President, Tokyo Metropolitan University, Meguro-Ku, 

Tokyo, Japan 
DANIELLI, DR. JAMES F., Department of Medicinal Chemistry, University of 

Buffalo School of Pharmacy, Buffalo, New York 14214 
DAVIS, DR. BERNARD D., Harvard Medical School, 25 Shattuck Street, Boston, 

Massachusetts 02115 
DAW, DR. NIGEL W., Department of Physiology, Washington University Medical 

School, 660 South Euclid Avenue, St. Louis, Missouri 63110 
DEHAAN, DR. ROBERT L., Department of Embryology, Carnegie Institution of 

Washington, Baltimore, Maryland 21210 
DELoRENZO, DR. ANTHONY, Anatomical and Pathological Research Laboratories, 

The Johns Hopkins Hospital, Baltimore, Maryland 21205 
DEPHILLIPS, DR. HENRY A., JR., Department of Chemistry, Trinity College, 

Hartford, Connecticut 06106 
DETTBARN, DR. WOLF-DEITRICH, Department of Pharmacology, Vanderbilt 

University, School of Medicine, Nashville, Tennessee 37217 

DEViLLAFRANCA, DR. GEORGE W., Department of Zoology, Smith College, North- 
ampton, Massachusetts 01060 
DIEHL, DR. FRED ALISON, Department of Biology, University of Virginia, Char- 

lottesville, Virginia 22903 
DOOLITTLE, DR. R. F., Department of Biology, University of California, La Jolla, 

California 92037 
DOWLING, DR. JOHN E., Biological Laboratories, Harvard University, 16 Divinity 

Avenue, Cambridge, Massachusetts 02138 

DRESDEN, DR. MARC II., Department of Biochemistry, Baylor College of Medi- 
cine, Houston, Texas 77025 
DUDLEY, DR. PATRICIA L., Department of Biological Sciences, Barnard College, 

Columbia University, New York, Ne\v York 10027 
DUNHAM, DR. PHILIP B., Department of Biology, Syracuse University, Syracuse, 

New York 13210 
DURYEE, DR. WILLIAM R., 3241 North Woodrow Street, Arlington, Virginia 

22207 
EBERT, DR. JAMES DAVID, Department, of Embryology, Carnegie Institution of 

Washington, Baltimore, Maryland 21210 
ECCLES, DR. JOHN C., Department of Biophysics and Physiology, State University 

of New York at Buffalo, Buffalo, New York 14214 " 
ECKERT, DR. ROGER O., Department of Zoology, University of California, 

Los Angeles, California 90024 
EDDS, DR. MAC V., JR., South College, University of Massachusetts, Amherst, 

Massachusetts 01002 
EDER, DR. HOWARD A., Albert Einstein College of Medicine, Bronx, New York 

10461 
EDWARDS, DR. CHARLES, Department of Biological Sciences, State University 

of New York at Albany, Albany, New York 12203 
EGYUD, DR. LASZLO G., The Institute for Muscle Research, Marine Biological 

Laboratory, Woods Hole, Massachusetts 02543 
EHRENSTEIN, DR. GERALD, National Institutes of Health, Bethesda, Maryland 

20014 



62 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

EICHEL, DR. HKRBERT J., Department of Biochemistry, Hahnemann Medical 

College, Philadelphia Pennsylvania 19102 
EISEN, DR. ARTHUR Z., Division of Dermatology, Washington University, School 

of Medicine, St. Louis, Missouri 63130 
EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis, 

Missouri 63130 
ELDER, DR. HUGH YOUNG, Institute of Physiology, University of Glasgow 

Glasgow, Scotland, U. K. 
ELLIOTT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann 

Arbor, Michigan 48104 
ELLIOTT, DR. GERALD F., Walton Hall, Bletchley, Bucks, The Open University, 

London, England, U. K. 
EPSTEIN, DR. HERMAN T., Department of Biology, Brandeis University, 

Waltham, Massachusetts 02154 

ERULKAR, DR. SOLOMON D., Department of Pharmacology, University of Penn- 
sylvania Medical School, Philadelphia, Pennsylvania 19104 
ESSNER, DR. EDWARD S., Sloan-Kettering Institute for Cancer Research, 410 E. 

68th Street, New York, New York 10021 
EVANS, DR. TITUS C., State University of Iowa, Radiation Research Laboratory, 

College of Medicine, Iowa City, Iowa 52240 
FAILLA, DR. P. M., Radiological Physics Division, Argonne National Laboratory, 

Argonne, Illinois 60439 

FARMANFARMAIAN, DR. ALLAHVERDI, Department of Physiology and Biochem- 
istry, Rutgers University, New Brunswick, New Jersey 08903 
FAURE-FREMIET, DR. EMMANUEL, College de France, Place M., Berthelot, Paris, 

France 
FAUST, DR. ROBERT GILBERT, Department of Physiology, University of North 

Carolina Medical School, Chapel Hill, North Carolina 27514 
FAWCETT, DR. D. W., Department of Anatomy, Harvard Medical School, 

Boston, Massachusetts 02115 
FERGUSON, DR. E. P., National Institute of General Medical Sciences, National 

Institutes of Health, Bethesda, Maryland 20014 
FERGUSON, DR. JAMES K. W ; ., Connought Laboratories, University of Toronto, 

Toronto 5, Ontario, Canada 
FIGGE, DR. F. H. J., University of Maryland Medical School, Lombard and Green 

Streets, Baltimore, Maryland 21201 

FINE, DR. JACOB, 8 Wolcott Road Ext., Chestnut Hill, Massachusetts 02167 
FINGERMAN, DR. MILTON, Department of Biology, Tulane LTniversity, New 

Orleans, Louisiana 70118 

FISCHER, DR. ERNST, 3110 Manor Drive, Richmond, Virginia 23230 
FISHER, DR. FRANK M., JR., Department of Biology, Rice University, Houston, 

Texas 77001 
FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, 

Toronto 5, Ontario, Canada 

FISHMAN, DR. Louis, 143 North Grove Street, Valley Stream, New York 11580 
FISHMAN, DR. MORTON, Department of Biological Sciences, State University 

of New York at Albany, Albany, New York 12203 
FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois, 

Urbana, Illinois 61801 



REPORT OF THE DIRECTOR 63 

FREEMAN, DR. ALAN RICHARD, Department of Physiology, Rutgers Medical 

School, New Brunswick, New Jersey 08903 
FREYGANG, DR. WALTER H., JR., 6247 29th Street, N. W., Washington, D. C. 

20015 

FRIES, DR. ERIK F. B., P. O. Box 605, Woods Hole, Massachusetts 02543 
FULTON, DR. CHANDLER M., Department of Biology, Brandeis University 

Waltham, Massachusetts 02154 
FUORTES, DR. MICHAEL G. F., National Institute for Neurological Diseases and 

Stroke, National Institutes of Health, Bethesda, Maryland 20014 
FURSHPAN, DR. EDWIN J., Department of Neurophysiology, Harvard Medical 

School, Boston, Massachusetts 02115 

FURTH, DR. JACOB, 99 Fort Washington Avenue, New York, New York 10032 
FYE, DR. PAUL M., Woods Hole Oceanographic Institution, Woods Hole, Massa- 
chusetts 02543 
GABRIEL, DR. MORDECAI L., Department of Biology, Brooklyn College, Brooklyn, 

New York 11210 
GAFFRON, DR. HANS, Department of Biology, Institute of Molecular Biophysics, 

Conradi Building, Florida State University, Tallahassee, Florida 32306 
GALL, DR. JOSEPH G., Department of Biology, Yale University, New Haven, 

Connecticut 06520 

GALTSOFF, DR. PAUL S., National Marine Fisheries Service, Woods Hole, Massa- 
chusetts 02543 
GELFANT, DR. SEYMOUR, Department of Dermatology, Medical College of 

Georgia, Augusta, Georgia 30904 
GELPERIN, DR. ALAN, Department of Biology, Princeton University, Princeton, 

New Jersey 08540 
GERMAN, DR. JAMES L., Ill, The New York Blood Center, 310 East 67th Street, 

New York, New York 10021 
GIBBS, DR. MARTIN, Department of Biology, Brandeis University, Waltham, 

Massachusetts 02154 

GIFFORD, DR. PROSSER, 97 Springs Street, Amherst, Massachusetts 01003 
GILBERT, DR. DANIEL L., Laboratory of Biophysics, NINDS, National Institutes 

of Health, Building 36, Room 2A-31, Bethesda, Maryland 20014 
GILMAN, DR. LAUREN C., Department of Biology, University of Miami, Coral 

Gables, Florida 33146 

GINSBERG, DR. HAROLD S., Department of Microbiology, University of Pennsyl- 
vania School of Medicine, Philadelphia, Pennsylvania 19104 
GIUDICE, DR. GIOVANNI, University of Palermo, Via Archirafi 22, Palermo, Italy 
GOLDEN, MR. WILLIAM T., 40 Wall Street, New York, New York 10005 
GOLDSMITH, DR. TIMOTHY H., Department of Biology, Yale University, New 

Haven, Connecticut 06520 
GOOCH, DR. JAMES L., Department of Biology, Juniata College, Huntingdon, 

Pennsylvania 16652 
GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, 

Atlanta, Georgia 30322 
GORMAN, DR. ANTHONY L. F., Laboratory of Neuropharmacology, SMI I, IRP, 

NIMH, St. Elizabeths Hospital, Washington, D. C. 20032 
GOTTSCHALL, DR. GERTRUDE Y., 315 East 68th Street, Apartment 9M, New 

York, New York 10021 



64 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

GRAHAM, DR. HERBERT, National Marine Fisheries Service, Woods Hole, Massa- 

chussetts 02543 

GRANT, DR. DAVID C., Box 2316, Davidson, North Carolina 28036 
GRANT, DR. PHILIP, Department of Biology, University of Oregon, Eugene, 

Oregon 97403 

GRASS, MR. ALBERT, The Grass Foundation, 77 Reservoir Road, Quincy, Massa- 
chusetts 02170 
GRASS, MRS. ELLEN R., The Grass Foundation, 77 Reservoir Road, Quincy, 

Massachusetts 02170 
GRAY, DR. IRVING E., Department of Zoology, Duke University, Durham, North 

Carolina 27706 
GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New 

Brunswick, New Jersey 08903 
GREEN, DR. JONATHAN P., School of Biological Sciences, University of Malaya, 

Kuala Lumpur, Malaysia 

GREEN, DR. MAURICE, Department of Microbiology, St. Louis University Medi- 
cal School, St. Louis, Missouri 63103 
GREENBERG, DR. MICHAEL J., Department of Biological Sciences, Florida State 

University, Tallahassee, Florida 32306 
GREGG, DR. JAMES H., Department of Zoology, University of Florida, Gainesville, 

Florida 32601 
GREGG, DR. JOHN R., Department of Zoology, Duke University, Durham, North 

Carolina 27706 
GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical 

College, New York, New York 10021 
GRIFFIN, DR. DONALD R., The Rockefeller University, 66 Street and York Avenue 

New York, New York 10021 
GROSCH, DR. DANIEL S., Department of Genetics, Garden Hall, North Carolina 

State University, Raleigh, North Carolina 27607 
GROSS, DR. JEROME, Developmental Biology Laboratory, Massachusetts General 

Hospital, Boston, Massachusetts 02114 

GROSS, DR. PAUL R., Department of Biology, Massachusetts Institute of Tech- 
nology, Cambridge, Massachusetts 02139 
GROSSMAN, DR. ALBERT, New York University Medical School, New York, New 

York 10016 
GRUNDFEST, DR. HARRY, Department of Neurology, Columbia University, 

College of Physicians and Surgeons, New York, New York 10032 
GUTTMAN, DR. RITA, Department of Biology, Brooklyn College, Brooklyn, New 

York 11210 
GWILLIAM, DR. G. F., Department of Biology, Reed College, Portland, Oregon 

97202 

HAJDU, DR. STEPHEN, National Institutes of Health, Bethesda, Maryland 20014 
HALVORSON, DR. HARLYN O., Department of Biology, Brandeis University, 

Waltham, Massachusetts 02154 
HAMILTON, DR. HOWARD L., Department of Biology, University of Virginia, 

Charlottesville, Virginia 22903 

HARDING, DR. CLIFFORD V., JR., Oakland University, Rochester, Michigan 48063 
HARRINGTON, DR. GLENN W., 11005 Jones Drive, Apt. 2, Parkville, Missouri 

64152 



REPORT OF THE DIRECTOR (o 

HARTLINE, DR. H. KEFFER, The Rockefeller University, New York, New York 
10021 

HARTMAN, DR. H. BERNARD, Department of Zoology, University of Iowa, Iowa 
City, Iowa 52240 

HARTMAN, DR. P. E., Department of Biology, The Johns Hopkins University, 
Baltimore, Maryland 21218 

HASCHEMEYER, DR. AUDREY E. V., Department of Biological Sciences, Hunter 
College, 695 Park Avenue, New York, New York 10021 

HASTINGS, DR. J. WOODLAND, Biological Laboratories, Harvard I'niversity, 
Cambridge, Massachusetts 02138 

HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo, 
New York 14203 

HAXO, DR. FRANCIS T., Department of Marine Botany, Scripps Institution of 
Oceanography, University of California, La Jolla, California 92038 

HAYASHI, DR. TERU, Department of Biology, Illinois Institute of Technology, 
Chicago, Illinois 60616 

HAYES, DR. RAYMOND L., JR., Department of Anatomy and Cell Biology, Univer- 
sity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 

HEGYELI, DR. ANDREW P., 8018 Aberdeen Road, Bethesda, Maryland 20014 

HENDLEY, DR. CHARLES 1)., 615 South Avenue, Highland Park, New Jersey 08904 

HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina, 
Chapel Hill, North Carolina 27514 

HERNDON, DR. WALTER R., Office of the Dean, College of Liberal Arts, 110 
Administration Building, University of Tennessee, Knoxville, Tennessee 37916 

HERVEY, MR. JOHN P., Box 85, Woods Hole, Massachusetts 02543 

HESSLER, DR. ANITA Y., 5795 Waverly Avenue, La Jolla, California 92037 

HIATT, DR. HOWARD H., Beth Israel Hospital, 330 Brookline Avenue, Boston, 
Massachusetts 02215 

HILL, DR. ROBERT BENJAMIN, Department of Zoology, University of Rhode 
Island, Kingston, Rhode Island 02881 

HILLMAN, DR. PETER, Department of Biology, Hebrew University, Jerusalem 

HINEGARDER, DR. RALPH T., Division of Natural Sciences, University of Cali- 
fornia, Santa Cruz, California 95060 

HINSCH, DR. GERTRUDE W., Institute of Molecular Evolution, 521 Anastasia, 
University of Miami, Coral Gables, Florida 33134 

HIRSHFIELD, DR. HENRY L., Department of Biology, Washington Square Center, 
New York University, New York, New York 10003 

HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge, 
Massachusetts 02138 

HODGE, DR. CHARLES, IV, Department of Biology, Temple University, Philadel- 
phia, Pennsylvania 19122 

HOFFMAN, DR. JOSEPH, Department of Physiology, Yale University School of 
Medicine, New Haven, Connecticut 06515 

HOLTZMAN, DR. ERIC, Department of Biological Science, Columbia University, 
New York, New York 10032 

HOLZ, DR. GEORGE G., JR., Department of Microbiology, State University of 
New York, Upstate Medical Center, Syracuse, New York 13210 

HOSKIN, DR. FRANCIS C. G., Biology Department, Illinois Institute of Tech- 
nology, Chicago, Illinois 60616 



66 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

HOSTETLER, DR. KARL V., Department of Medicine, Case Western Reserve 

University, Cleveland, Ohio 44106 

HOUSTON, MR. HOWARD, Preston Avenue, Meriden, Connecticut 06450 
HUMES, DR. ARTHUR G., Systematics-Ecology Program, Marine Biological 

Laboratory, Woods Hole, Massachusetts 02543 
HUMPHREYS, DR. TOM DANIEL, Department of Biology, University of California, 

San Diego, La Jolla, California 92037 
HUNTER, DR. BRICK, Department of Zoology, University of Rhode Island, 

Kingston, Rhode Island 02881 
HURWITZ, DR. CHARLES, Basic Science Research Laboratory, VA Hospital, 

Albany, New York 12208 
HURWITZ, DR. JERARD, Department of Molecular Biology, Albert Einstein College 

of Medicine, Bronx, New York 10461 
HUXLEY, DR. HUGH E., Medical Research Council, Laboratory of Molecular 

Biology, Cambridge, England, U. K. 
HYDE, DR. BEAL B., Department of Botany, University of Vermont, Burlington, 

Vermont 05401 

HYDE, MR. ROBINSON, Montgomery Road, RR 2, Skillman, New Jersey 08558 
ILAN, DR. JOSEPH, Department of Anatomy, Case Western Reserve University, 

School of Medicine, Cleveland, Ohio 44106 
INOUE, DR. SADAYUKI, Department of Biochemistry, University of Montreal, 

Montreal, P. Q., Canada 
INOUE, DR. SHINYA, 217 Leidy Building, Department of Biology, University of 

Pennsylvania, Philadelphia, Pennsylvania 19104 
ISENBERG, DR. IRVIN, Science Research Institute, Oregon State University, 

Corvallis, Oregon 97330 

ISSELBACHER, DR. KURT (., Massachusetts General Hospital, Boston, Massa- 
chusetts 02114 
JACOBSON, DR. AN TOM-; ( '.., Department of Biology, University of Texas, Austin, 

Texas 78710 
JAFFE, LIONEL, Department of Biology, Purdue University, Lafayette, Indiana 

46207 
JANNASCH, DR. HOLGER W., \Voods Hole Oceanographic Institution, Woods 

Hole, Massachusetts 02543 
JANOFF, DR. AARON, Department of Pathology, New York University School of 

Medicine, 550 First Avenue, New York, New York 10016 
JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina, 

Chapel Hill, North Carolina 27514 
JENNINGS, DR. JOSEPH B., Department of Zoology, University of Leeds, Leeds 

LS2 9JT, England, U. K. 

JOHNSON, DR. FRANK H., Department of Biology, Princeton University, Prince- 
ton, New Jersey 08540 
JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of 

Florida, Gainesville, Florida 32601 
JONES, DR. MEREDITH L., Division of Worms, Museum of Natural History, 

Smithsonian Institution, Washington, D. C. 20650 
JONES, DR. RAYMOND F., Department of Biology, State University of New York 

at Stony Brook, Long Island, New York 11753 



REPORT OF THE DIRECTOR 67 

JOSEPHSON, DR. R. K., School of Biological Sciences, University of California, 
Irvine, California 92664 

KAAN, DR. HELEN W., Box 665, Woods Hole, Massachusetts 02543 

KABAT, DR. E. A., Neurological Institute, Columbia University, College of 
Physicians and Surgeons, New York, New York 10032 

KALEY, DR. C.ABOR, New York Medical College, Flower and Fifth Avenue Hos- 
pital, 5th Avenue at 106th Street, New York, New York 10029 

KAMINER, DR. BENJAMIN, Department of Physiology, Boston University School 
of Medicine, Boston, Massachusetts 02118 

KANE, DR. ROBERT F., Pacific Biomedical Research Center, 2538 The Mall, 
University of Hawaii, Honolulu, Hawaii 96822 

KARAKASHIAN, DR. STEPHEN J., P. O. Box 210, Old Westbury, New York 11568 

KARUSH, DR. FRED, Department of Microbiology, University of Pennsylvania 
School of Medicine, Philadelphia, Pennsylvania 19104 

KATZ, DR. GEORGE M., Department of Neurology, Columbia University, College 
of Physicians and Surgeons, 630 West 168th Street, New York, New York 
10032 

KAUFMAN, DR. B. P., Department of Zoology, University of Michigan, Ann 
Arbor, Michigan 48104 

KEAN, DR. EDWARD L., Departments of Chemistry and Ophthalmology, Case 
Western Reserve University, Cleveland, Ohio 44101 

KELLEY, ROBERT E., Department of Anatomy and Psychology, Dartmouth 
Medical School, Hanover, New Hampshire 03755 

KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann 
Arbor, Michigan 48104 

KEMPTON, DR. Ri DOLE T., 924 Shore Drive, St. Augustine, Florida 32084 

KEOSIAN, DR. JOHN, 18 Meadow Lane, Amity ville, New York 11710 

KETCHUM, DR. BOSTWICK H., Woods Hole Oceanographic Institution, Woods 
Hole, Massachusetts 02543 

KEYNAN, DR. ALEXANDER, Institute for Biological Research, Ness-Ziona, Israel 

KILLE, DR. FRANK R., 340 Albany Shaker Road, Londonville, New York 12211 

KIND, DR. C. ALBERT, Department of Zoology, University of Connecticut, Storrs, 
Connecticut 06268 

KINDRED, DR. JAMES E., 2010 Hessian Road, Charlottesville, Virginia 22903 

KING, DR. Thomas J., Georgetown University, Department of Biology, Washing- 
ton, D. C. 20007 

KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca, 
New York 14850 

KLEIN, DR. MORTON, Department of Microbiology, Temple University, Phila- 
delphia, Pennsylvania 19122 

KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland, 
Oregon 97202 

KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evans- 
ton, Illinois 60201 

KOHLER, KURT, Department of Biochimie Macromoleculaire, C.N.R.S., Uni- 
versite de Montpellier, Montpellier, France 

KONIGSBERG, DR. IRWIN R., Department of Biology, Gilmer Hall, University of 
Virginia, Charlottesville, Virginia 22903 



68 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

KORR, DR. I. M., Department of Physiology, Kirksville College of Osteopathy, 

Kirksville, Missouri 63501 
KRAHL, DR. M. E., Department of Physiology, Stanford University, Stanford, 

California 

KRANE, DR. STEPHEN M., Massachusetts General Hospital, Boston, Massa- 
chusetts 02114 

KRASSNER, DR. STUART MITCHELL, Department of Organismic Biology, Uni- 
versity of California, Irvine, California 92650 

KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Balti- 
more, Maryland 21201 
KREIG, DR. WENDELL J. S., Northwestern Medical School, 303 East Chicago 

Avenue, Chicago, Illinois 60611 
KRIEBEL, DR. MAHLON E., Department of Physiology, State University of New 

York, Upstate Medical Center, Syracuse, New York 13210 
KRUPA, DR. PAUL L., Department of Biology, The City College of New York, 

139th St., and Convent Avenue, New York, New York 10031 
KUFFLER, DR. STEPHEN W., Department of Neurophysiology, Harvard Medical 

School, Boston, Massachusetts 02115 
KUNITZ, DR. MOSES, The Rockefeller University, 66th Street and York Avenue, 

New York, New York 10021 
KUSANO, DR. KIYOSHI, Biology Department, Illinois Institute of Technology, 

3300 Federal Street, Chicago, Illinois 61606 

LAMARCHE, DR. PAUL H., 593 Eddy St., Providence, Rhode Island 02903 
LAMY, DR. FRANCOIS, Department of Biochemistry, University of Sherbrooke, 

School of Medicine, Sherbrooke, Quebec, Canada 
LANCEFIELD, DR. D. E., 203 Arleigh Road, Douglaston, Long Island, New York 

11363 
LANCEFIELD, DR. REBECCA C., The Rockefeller University, 66th Street and York 

Avenue, New York, New York 10021 
LANDIS, DR. E. M., Department of Biology, Williams Hall, Lehigh University, 

Bethlehem, Pennsylvania 18015 
LANSING, DR. ALBERT, L, Department of Anatomy, University of Pittsburgh 

School of Medicine, Pittsburgh, Pennsylvania 15213 
LASH, DR. JAMES W., Department of Anatomy, University of Pennsylvania 

School of Medicine, Philadelphia, Pennsylvania 19104 
LASTER, DR. LEONARD, National Institute of Arthritis and Metabolic Diseases, 

National Institutes of Health, Bethesda, Maryland 20014 
LAUFER, DR. HANS, Department of Zoology and Entomology, University of 

Connecticut, Storrs, Connecticut 06268 
LAUFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, 

Pittsburgh, Pennsylvania 15213 

LAVIN, DR. GEORGE L, 6200 Norvo Road, Baltimore, Maryland 21207 
LAWLER, DR. H. CLAIRE, 336 West 246th Street, Riverdale, New York 10471 
LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota 

Medical School, Minneapolis, Minnesota 55455 
LEADBETTER, DR. EDWARD R., Department of Biology, Amherst College, Am- 

herst, Massachusetts 01002 
LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford Medical School, 

Palo Alto, California 94304 



REPORT OF THE DIRECTOR 69 

LEE, DR. RICHARD E., Cornell University College of Medicine, New York, New 

York 10021 
LEFEVRE, DR. PAUL G., Department of Physiology, State University of New 

York at Stony Brook, Stony Brook, New York 11790 

LENHER, DR. SAMUEL, 1900 Woodlawn Avenue, Wilmington, Delaware 19806 
LERMAN, DR. SIDNEY, Mclntyre Medical Science Center, McGill University, 

Room 12H, Montreal, Canada 

LERNER, DR. AARON B., Yale Medical School, New Haven, Connecticut 06515 
LEVIN, DR. JACK, Department of Medicine, The Johns Hopkins Hospital, 

Baltimore, Maryland 21205 

LEVINE, DR. RACHMIEL, City of Hope Medical Center, Duarte, California 91010 
LEVINTHAL, DR. CYRUS, Department of Biological Sciences, Columbia University, 

908 Schermerhorn Hill, New York, New York 10027 

LEVY, DR. MILTON, 39-95-48th Street, Long Island City, New York 11104 
LEWIN, DR. RALPH A., Scripps Institution of Oceanography, La Jolla, California 

92037 
LEWIS, DR. HERMAN W., Genetic Biology Program, National Science Foundation, 

Washington, D. C. 20025 

LING, DR. GILBERT, 307 Berkeley Road, Merion, Pennsylvania 19066 
LINSKENS, DR. H. P., Department of Botany, University of Driehuizenveg 200, 

Nijmegen, The Netherlands 
LIPICKY, DR. RAYMOND J., Department of Pharmacology, College of Medicine, 

University of Cincinnati, Eden and Bethesda Avenues, Cincinnati, Ohio 45219 
LITTLE, DR. E. P., 216 Highland Street, West Newton, Massachusetts 02158 
LIUZZI, DR. ANTHONY, Division of Nuclear Science, Lowell Technological Insti- 
tute, Lowell, Massachusetts 01854 

LLOYD, DR. DAVID P. C., The Rockefeller University, New York, New York 10021 
LOCHHEAD, DR. JOHN H., Department of Zoology, Life Sciences Building, 

University of Vermont, Burlington, Vermont 05401 
LOEB, DR. R. F., 950 Park Avenue, New York, New York 10028 
LOWENSTEIN, DR. WERNER R., Physiology and Biophysics, School of Medicine, 

University of Miami, P. O. Box 875, Miami, Florida 33152 
LOEWUS, DR. FRANK A., Department of Biology, State University of New York 

at Buffalo, Buffalo, New York 14214 
LOFTFIELD, DR. ROBERT B., Department of Biochemistry, University of New 

Mexico Medical School, Albuquerque, New Mexico 87106 
LONDON, DR. IRVING M., Department of Medicine, Albert Einstein College of 

Medicine, New York, New York 10461 
LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, 

Evanston, Illinois 60201 
LOVE, DR. WARNER E., Department of Biophysics, Johns Hopkins University, 

Baltimore, Maryland 21218 
LUBIN, DR. MARTIN, Department of Microbiology, Dartmouth Medical School, 

Hanover, New Hampshire 03755 
LURIA, DR. SALVADOR E., Department of Biology, Massachusetts Institute of 

Technology, Cambridge, Massachusetts 02139 

LYNCH, DR. CLARA J., 4800 Fillmore Avenue, Alexandria, Virginia 22311 
LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America, 

Washington, D. C. 20017 



70 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

MAcNiCHOL, EDWARD F., JR., National Institutes of Health, Bldg. 31 Room 

SA-52, Bethesda, Maryland 20014 

MAGRUDER, DR. SAMUEL R., Route 4, Box 177, Kevil, Kentucky 42053 
MAHLER, DR. HENRY R., Department of Biochemistry, Indiana University, 

Bloomington, Indiana 47401 
MALKIEL, DR. SAUL, Children's Cancer Research Foundation, Inc., 35 Binney 

Street, Boston, Massachusetts 02115 
MANGUM, CHARLOTTE; P., Department of Biology, College of William and Mary, 

Williamsburg, Virginia 23185 
MARKS, DR. Paul A., Columbia University, College of Physicians and Surgeons, 

New York, Xew York 10032 
MARSH, DR. JULIAN B. Department of Biochemistry, University of Pennsylvania 

School of Dental Medicine, 4001 Spruce St., Philadelphia, Pennsylvania 19104 
MARSLAND, DR. DOUGLAS A., 48 Church Street, Woods Hole, Massachusetts 

02543 
MAUTNER, DR. HENRY C,., Tufts University School of Medicine, 136 Harrison 

Avenue, Department of Biochemistry and Pharmacy, Boston, Massachusetts 

02111 
MAZIA, DR. DANIEL, Department of Zoology, University of California, Berkeley, 

California 94720 
McCANN, DR. FRANCES, Department of Physiology, Dartmouth Medical School, 

Hanover, New Hampshire 03755 
\l< CLOSKEY, DR. LAWRENCE R., Department of Biology, Walla Walla College, 

College Place, Washington 99324 
McDANiEL, DR. JAMES SCOTT, Department of Biology, East Carolina College, 

Greenville, North Carolina 28734 
MCDONALD, SISTER ELIZABETH SETON, Department of Biology, College of Mt. 

St. Joseph on the Ohio, Mt. St. Joseph, Ohio 45051 
McELROY, DR. WILLIAM D., Office of the Director, National Science Foundation, 

Washington, D. C. 20550 
McREYNOLDS, DR. JOHN S., Laboratory of Neurophysiology, MINDB, National 

Institute of Health, Bethesda, Maryland 20014 
MEINKOTH, DR. NORMAN, Department of Biology, Swarthmore College, Swarth- 

more, Pennsylvania 19081 
MELLON, DR. DEFOREST, JR., Department of Biology, University of Virginia, 

Charlottesville, Virginia 22903 
MENDELSON, DR. MARTIN, Health Sciences Centers, State University of New 

York, Stony Brook, New York 11790 
METZ, DR. C. B., Institute of Molecular Evolution, University of Miami, Coral 

Gables, Florida 33146 

METZ, DR. CHARLES W., Box 174, Woods Hole, Massachusetts 02543 
MIDDLEBROOK, DR. ROBERT, Downsway, School Lane, Kirk Ella, Hull, England, 

U. K. HW10 7NR 
MILKMAN, DR. ROGER D., Department of Zoology, University of Iowa, Iowa 

City, Iowa 52240 
MILLER, DR. J. A., JR., Department of Anatomy, Tulane University, New 

Orleans, Louisiana 70112 
MILLOTT, DR. NORMAN, Universities Marine Biological Station, Millport, Isle 

of Cumbrae, Scotland, U. K. 



REPORT OF THE DIRECTOR 71 

MILLS, DR. ERIC LEONARD, Institute of Oceanography, Dalhousie University, 

1 falifax, Nova Scotia, Canada 
MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire, 

Durham, New Hampshire 03824 
MONROY, DR. ALBERTO, CNR Laboratory of Molecular Embryology, 80072 

Arco Felice, Napoli, Italy 
MOORE, DR. JOHN A., Department of Life Sciences, University of California, 

Riverside, California 92502 
MOORE, DR. JOHN W., Department of Physiology, Duke University Medical 

Center, Durham, North Carolina 27706 
MOORE, DR. R. O., Associate Dean, College of Biological Sciences, Ohio State 

University, Columbus, Ohio 43210 
MORAN, DR. JOSEPH F., JR., Department of Biology, Sacred Heart University, 

Bridgeport, Connecticut 06604 
MORIN, DR. JAMES G., Department of Zoology, University of California, Los 

Angeles, California 90052 
MORLOCK, DR. NOEL, Department of Surgery, Detroit General Hospital, 1326 St. 

Antoine Street, Detroit, Michigan 48226 
MORRILL, DR. JOHN B., JR., Division of Natural Sciences, New College, Sarasota, 

Florida 33478 

MORSE, DR. RICHARD STETSON, 193 Winding River Road, Wellesley, Massa- 
chusetts 02184 
MOSCONA, DR. A. A., Department of Zoology, University of Chicago, Chicago, 

Illinois 60637 
MOTE, DR. MICHAEL I., Department of Biology, Temple University, Philadelphia, 

Pennsylvania 19122 
MOIL, DR. E. T., Department of Biology, Rutgers University, New Brunswick, 

New Jersey 08903 

MOUNTAIN, DR. ISABEL M., 2 Lilac Place, Thornwood, New York 10594 
MILLINS, Dr. LORIN J., Department of Biophysics, University of Maryland 

School of Medicine, Baltimore, Maryland 21201 

AlrsACCHiA, DR. XAVIER J., Department of Physiology, Medical Center, Uni- 
versity of Missouri, Columbia, Missouri 65201 

NABRIT, DR. S. M., 686 Beckwith Street, S. W. Atlanta, Georgia 30314 
NACE, DR. PAUL FOLEY, 3310A Fall Creeway East, Indianapolis, Indiana 46205 
NACHMANSOHN, DR. DAVID, Department of Neurology, Columbia University, 

College of Physicians and Surgeons, New York, New York 10032 
NARAHASHI, DR. TOSHIO, Department of Physiology, Duke University Medical 

Center, Durham, North Carolina 27706 
NASATIR, DR. MAIMON, Department of Biology, University of Toledo, Toledo, 

Ohio 43606 
NASON, DR. ALVIN, McCollum-Pratt Institute, The Johns Hopkins L T niversity, 

Baltimore, Maryland 21218 
NELSON, DR. LEONARD, Department of Physiology, Medical College of Ohio at 

Toledo, Toledo, Ohio 43614 
NEURATH, DR. H., Department of Biochemistry, University of Washington, 

Seattle, Washington 98105 
NICHOLLS, DR. JOHN GRAHAM, Department of Neurobiology, Harvard Medical 

School, 25 Shattuck Street, Boston, Massachusetts 02115 



72 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

NICOLL, DR. PAUL A., R.R. 12, Box 286, Bloomington, Indiana 47401 
Niu, DR. MAN-CHIANG, Department of Biology, Temple University, Phila- 
delphia, Pennsylvania 19122 
NOVIKOFF, DR. ALEX B., Department of Pathology, Albert Einstein College of 

Medicine, Bronx, New York 10461 
NYSTROM, DR. RICHARD A., Department of Biological Sciences, University of 

Delaware, Newark, Delaware 19711 
OCHOA, DR. SEVERO, New York University College of Medicine, New York, New 

York 10016 
ODUM, DR. EUGENE, Department of Zoology, University of Georgia, Athens, 

Georgia 30601 

OLSON, DR. JOHN M., Brookhaven National Laboratory, Upton, New York 11973 
OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn 

Mawr, Pennsylvania 19010 

OSTERHOUT, DR. MARION IRWIN, 160 E. 65th Street, New York, New York 10021 
PALMER, DR. JOHN D., Department of Biology, New York University, University 

Heights, New York, New York 10053 
PALTI, DR. YORAM, Hebrew University School of Medicine, Department of 

Physiology, Box 1172, Jerusalem, Israel 
PAPPAS, DR. GEORGE D., Department of Anatomy, Albert Einstein College of 

Medicine, Bronx, New York 10461 
PARNAS, DR. ITZCHAK, Department of Zoology, Hebrew University, Jerusalem, 

Israel 
PASSANO, DR. LEONARD M., Department of Zoology, University of Wisconsin, 

Madison, Wisconsin 53706 
PEARLMAN, DR. ALAN L., Department of Physiology, Washington University 

School of Medicine, St. Louis, Missouri 63110 
PERSON, DR. PHILIP, Special Dental Research Program, Veterans Administration 

Hospital, Brooklyn, New York 11219 
PETTIBONE, DR. MARIAN H., Division of Marine Invertebrates, U. S. National 

Museum, Washington, D. C. 20025 
PFOHL, DR. RONALD J., Department of Zoology, Miami University, Oxford, 

Ohio 45056 
PHILPOTT, DR. DELBERT, E., MASA Ames Research Center, Moffett Field, 

California 94035 

POLLACK, DR. LELAND W., 59 School Street, Woods Hole, Massachusetts 02543 
PORTER, DR. KEITH R., 748 llth Street, Boulder, Colorado 80302 
POTTER, DR. DAVID, Department of Neurophysiology, Harvard Medical School, 

Boston, Massachusetts 02115 
POTTS, DR. WILLIAM T. W., Department of Biology, University of Lancaster, 

Lancaster, England, U. K. 
PRENDERGAST, DR. ROBERT A., Department of Pathology and Opthalmology, 

Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 
PRICE, DR. CARL A., Department of Biochemistry and Microbiology, Rutgers 

University, New Brunswick, New Jersey 08803 
PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, 

Baltimore, Maryland 21212 
PROSSER, DR. C. LADD, Department of Physiology and Biophysics, Burrill Hall, 

University of Illinois, Urbana, Illinois 61803 



REPORT OF THE DIRECTOR 73 

PROVASOLI, DR. LUIGI, Haskins Laboratories, 165 Prospect Street, New Haven, 

Connecticut 06520 
RABIN, DR. HARVEY, Director, Department of Virology and Cell Biology, 

Bionetics Research Laboratories, 5510 Nicholson Lane, Kensington, Maryland 

20795 
RAMSEY, DR. ROBERT W., Department of Physiology, Medical College of Virginia, 

Richmond, Virginia 23150 
RANKIN, DR. JOHN S., Department of Zoology, University of Connecticut, Storrs, 

Connecticut 06268 
RANZI, DR. SILVIO, Department of Zoology, University of Milan, Via Celonia 10, 

Milan, Italy 
RAPPORT, DR. M., Department of Pharmacology, Columbia University, College 

of Physicians and Surgeons, New York, New York 10032 
RATNER, DR. SARAH, Department of Biochemistry, The Public Health Research 

Institute of the City of New York, Inc., 455 First Avenue, New York, New 

York 10016 
RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta, 

Georgia 30322 
READ, DR. CLARK P., Department of Biology, Rice University, Houston, Texas 

77001 
REBHUN, DR. LIONEL I., Department of Biology, Gilmer Hall, University of 

Virginia, Charlottesville, Virginia 22901 

RECKNAGEL, DR. R. O., Department of Physiology, Case Western Reserve Uni- 
versity, Cleveland, Ohio 44106 

REDFIELD, DR. ALFRED C., Maury Lane, Woods Hole, Massachusetts 02543 
REINER, DR. JOHN M., Department of Biochemistry, Albany Medical College 

of Union University, Albany, New York 12208 
RENN, DR. CHARLES E., Route 2, Hampstead, Maryland 21074 
REUBEN, DR. JOHN P., Department of Neurology, Columbia University, College 

of Physicians and Surgeons, Xe\v York, New York 10032 
REYNOLDS, DR. GEORGE THOMAS, Palmer Laboratory, Princeton University, 

Princeton, New Jersey 08540 

REZNIKOFF, DR. PAUL, 151 Sparks Ave., Pelham, New York 10803 
RHOADS, DR. DONALD C., Department of Geology-Geophysics, Yale University, 

New Haven, Connecticut 06510 
RICE, DR. ROBERT VERNON, Mellon Institute, Carnegie-Mellon University, 4400 

Fifth Avenue, Pittsburgh, Pennsylvania 15213 
RICH, DR. ALEXANDER, Department of Biology, Massachusetts Institute of 

Technology, Cambridge, Massachusetts 02139 

RICHARDS, DR. A., 2950 East Marble Street, Tucson, Arizona 85716 
RICHARDS, DR. A. GLENN, Department of Entomology, University of Minnesota, 

St. Paul, Minnesota 55101 
RICHARDS, DR. OSCAR W., Pacific University, College of Optometry, Forrest 

Grove, Oregon 97116 
RIPPS, DR. HARRIS, Department of Opthalmology, New York University, School 

of Medicine, 550 1st Avenue, New York, New York 10016 
ROBERTS, DR. JOHN L., Department of Zoology, University of Massachusetts, 

Amherst, Massachusetts 01002 
ROBINSON, DR. DENIS M., 19 Orlando Avenue, Arlington, Massachusetts 02174 



74 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

ROCKSTEIN, DR. MORRIS, Department of Physiology, University of Miami 

School of Medicine, P.O. Box 875 Biscayne Annex, Miami, Florida 33152 
ROMER, DR. ALFRED S., Museum of Comparative Zoology, Harvard University, 

Cambridge, Massachusetts 02138 
RONKIN, DR. RAPHAEL E., National Science Foundation, O.I.S.A., Washington, 

D. C. 20550 

ROOT, DR. W. S., 20 Brooks Road, Woods Hole, Massachusetts 02543 
ROSE, DR. S. MERYL, Laboratory of Developmental Biology, Tulane University, 

F. Edward Hebert Center, Belle Chasse, Louisiana 70037 
ROSENBERG, DR. EVELYN K., Jersey City State College, Jersey City, New Jersey 

07305 
ROSENBERG, DR. PHILIP, Division of Pharmacology, University of Connecticut, 

School of Pharmacy, Storrs, Connecticut 06268 

ROSENBLUTH, Miss RAJA, Kinsmen Laboratory for Neurological Research, Uni- 
versity of British Columbia, Vancouver 8, British Columbia, Canada 
ROSENKRANZ, DR. HERBERT S., Department of Microbiology, Columbia Uni- 
versity, College of Physicians and Surgeons, New York, New York 10032 
ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pitts- 
burgh Medical School, Pittsburgh, Pennsylvania 15213 
ROSLANSKY, DR. JOHN, 26 Albatross, Woods Hole, Massachusetts 02543 
ROTH, DR. JAY S., Division of Biological Sciences, Section of Biochemistry and 

Biophysics, University of Connecticut, Storrs, Connecticut 06268 
ROTHENBERG DR. M. A., Desert Test Center, Ft. Douglas, Salt Lake City, 

Utah 84113 
ROWLAND, DR. LEWIS P., Department of Neurology, Hospital of the University 

of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104 
RUBINOW, DR. SOL I., Cornell University, Medical College, Department of 

Biomathematics, New York, New York 1001 2 
RUGH, DR. ROBERTS, The Pavilion S306, 12,000 Old Georgetown Rd., Rockville, 

Maryland 20852 
RUSHFORTH, DR. NORMAN B., Department of Biology, Case Western Reserve 

University, Cleveland, Ohio 44106 
RUSSELL-HUNTER, DR. W. D., Department of Biology, Lyman Hall, Syracuse 

University, Syracuse, New York 13210 
RUSTAD, DR. RONALD C., Department of Radiology, Case Western Reserve 

University, Cleveland, Ohio 44106 
RUTMAN, DR. ROBERT J., University of Pennsylvania, School of Veterinary 

Medicine, Department of Animal Biology, 3800 Spruce Street, Philadelphia, 

Pennsylvania 19104 
RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole, 

Massachusetts 02543 
SAGER, DR. RUTH, Department of Biological Sciences, Hunter College, 695 Park 

Avenue, New York, New York 10021 
SANBORN, DR. RICHARD C., Dean, Purdue University Regional Campus, 1125 

East 38th Street, Indianapolis, Indiana 46205 
SANDERS, DR. HOWARD L., Woods Hole Oceanographic Institution, Woods Hole, 

Massachusetts 02543 
SATO, DR. HIDEMI, 217 Leidy Building, Department of Biology, University of 

Pennsylvania, Philadelphia, Pennsylvania 19104 



REPORT OF THE DIRECTOR 

SAUNDERS, DR. JOHN W., JR., Department of Biological Sciences, State University 

of New York, at Albany, Albany, New York 12203 
SAZ, DR. ARTHUR KENNETH, Department of Microbiology, Georgetown University 

Medical and Dental Schools, 3900 Reservoir Road, Washington, D. C. 20007 
SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of 

California, Berkeley, California 94720 
SCHARRER, DR. BERTA V., Department of Anatomy, Albert Einstein College of 

Medicine, Bronx, New York 10461 
SCHLESINGER, DR. R. WALTER, Department of Microbiology, Rutgers Medical 

School, New Brunswick, New Jersey 08903 
SCHMEER, SISTER ARLINE CATHERINE, O.P., Institutum Divi Life Sciences 

Laboratory, Ohio Dominican College, Columbus, Ohio 43219 
SCHMIDT, DR. L. H., Southern Research Institute, 2000 Ninth Avenue South, 

Birmingham, Alabama 35205 
SCHMITT, DR. FRANCIS O., Neurosciences Research Program, Massachusetts 

Institute of Technology, 280 Newton Street, Brookline, Massachusetts 02146 
SCHMITT, DR. O. H., University of Minnesota, 200 T.N.C.E., Minneapolis, 

Minnesota 55455 
SCHNEIDERMAN, DR. HOWARD A., Department of Organismic Biology, School of 

Biological Sciences, University of California, Irvine, California 92664 
SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, California 

92037 
SCHOPF, DR. THOMAS J. M., Department of the Geophysical Sciences, University 

of Chicago, 5734 S. Ellis Avenue, Chicago, Illinois 60637 
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, 

Massachusetts 01002 
SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington, 

Indiana 47401 
SCHUEL, DR. HERBERT, Anatomy Department, Mount Sinai School of Medicine, 

New York, New York 10029 
SCHUETZ, DR. ALLEN WALTER, The Johns Hopkins University School of Hygiene 

and Public Health, Baltimore, Maryland 21205 
SCHWARTZ, DR. TOBIAS L., Biological Sciences Group, University of Connecticut 

Storrs, Connecticut 06268 

SCOTT, DR. ALLAN C., Colby College, Waterville, Maine 02901 
SCOTT, Dr. GEORGE, T., Department of Biology, Oberlin College, Oberlin, Ohio 

44074 

SEARS, DR. MARY, Box 152, Woods Hole, Massachusetts 02543 
SELIGER, DR. HOWARD H., McCollum-Pratt Institute, The Johns Hopkins 

University, Baltimore, Maryland 21218 
SENFT, DR. ALFRED W., Department of Medical Sciences, Brown University, 

Providence, Rhode Island, 02912 
SENFT, DR. JOSEPH P., Department of Physiology, Rutgers University, New 

Brunswick, New Jersey 08903 
SHANKLIN, DR. DOUGLAS R., Pathologist-in-chief, University of Chicago, Chicago, 

Lying-in Hospital, Chicago, Illinois 60637 
SHAPIRO, DR. HERBERT, 6025 North 13th Street, Philadelphia, Pennsylvania 

19141 



76 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East 

Lansing, Michigan 48823 
SHEDLOVSKY, DR. THEODORE, The Rockefeller University, New York, New York 

10021 

SHEMIN, DR. DAVID, Department of Chemistry and Biological Sciences, North- 
western University, Evanston Illinois 60201 
SHEPRO, DR. DAVID, Department of Biology, Boston University, 2 Cummington 

Street, Boston, Massachusetts 02215 
SHERMAN, DR. I. W., Division of Life Sciences, University of California, Riverside, 

California 92502 
SHILO, DR. MOSHE, Head, Department of Microbiological Chemistry, Hebrew 

University, Jerusalem 
SICHEL, MRS. F. J. M., Department of Biology, Trinity College, Burlington, 

Vermont 05401 
SIEGEL, DR. IRWIN M., Department of Ophthalmology, New York University 

Medical Center, 550 First Avenue, New York, New York 10016 
SIEGELMAN, DR. HAROLD W., Department of Biology, Brookhaven National 

Laboratory, Upton, New York 11973 
SILVER, DR. PAUL C., Department of Botany, University of California, Berkeley, 

California 94720 
SIMMONS, DR. JOHN E., JR., Department of Biology, University of California, 

Berkeley, California 94720 
SIMON, DR. ERIC J., New York University Medical School, 550 First Avenue, 

New York, New York 10016 
SJODIN, DR. RAYMOND A., Department of Biophysics, University of Maryland 

School of Medicine, Baltimore, Maryland 21201 
SKINNER, DR. DOROTHY M., Biology Division, Oak Ridge National Laboratory, 

Oak Ridge, Tennessee 37830 

SLIFER, DR. ELEANOR H., 308 Lismore Avenue, Glenside, Pennsylvania 19038 
SLOBODKIN, DR. LAWRENCE BASIL, Department of Biology, State University of 

New York at Stony Brook, Stony Brook, New York 11790 
SMELSER, DR. GEORGE K., Department of Anatomy, Columbia University, New 

York, New York 10032 
SMITH, MR. HOMER P., General Manager, Marine Biological Laboratory, Woods 

Hole, Massachusetts 02543 

SMITH, MR. PAUL FERRIS, Church Street, Woods Hole, Massachusetts 02543 
SMITH, DR. RALPH I., Department of Zoology, Lhiiversity of California, Berkeley, 

California 94720 
SONNENBLICK, DR. B. P., Department of Biology, Rutgers University, 195 

University Avenue, Newark, New Jersey 07102 

SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Blooming- 
ton, Indiana 47401 
SPECTOR, DR. A., Department of Ophthalmology, Columbia University, College 

of Physicians and Surgeons, New York, New York 10032 
SPEIDEL, DR. CARL C., 1873 Field Road, Charlottesville, Virginia 22903 
SPIEGEL, DR. MELVIN, Department of Biological Sciences, Dartmouth College, 

Hanover, New Hampshire 03755 
SPINDEL, DR. WILLIAM, Belfer Graduate School of Science, Yeshiva University, 

Amsterdam Avenue and 186th Street, Bronx, New York 10461 



REPORT OF THE DIRECTOR 77 

SPIRTES, DR. MORRIS ALBERT, Veterans Administration Hospital, 1601 Perdido 
Street, New Orleans, Louisiana 70112 

SPRATT, DR. NELSON T., Department of Zoology, University of Minnesota, 
Minneapolis, Minnesota 55414 

STARR, DR. RICHARD C., Department of Botany, Indiana University, Blooming- 
ton, Indiana 47401 

STEINBACH, DR. H. BURR, Dean of Graduate Studies, Woods Hole Oceanographic 
Institution, Woods Hole, Massachusetts 02543 

STEINBERG, DR. MALCOLM S., Department of Biology, Princeton University, 
Princeton, New Jersey 08540 

STEINHARDT, DR. JACINTO, Science Advisor to the President, Georgetown Uni- 
versity, Washington, D. C. 20007 

STEPHENS, DR. GROVER C., Division of Biological Sciences, University of Cali- 
fornia, Irvine, California 92650 

STEPHENS, DR. RAYMOND E., Department of Biology, Brandeis University, 
Waltham, Massachusetts 02154 

STETTEN, DR. DEWITT, National Institute of General Medical Sciences, National 
Institutes of Health, Bethesda, Maryland 20014 

STETTEN, DR. MARJORIE R., Building 10, National Institutes of Health, Bethesda, 
Maryland 20014 

STRACHER, ALFRED, Downstate Medical Center, State University of New York 
at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York 11203 

STREHLER, DR. BERNARD L., 5184 Willoxv Wood Roeid, Rolling Hills Estate, 
California 90274 

STRITTMATTER, DR. PHILIPP, Department of Biochemistry, University of Con- 
necticut, School of Medicine, Health Center, Hartford Plaza, Hartford, 
Connecticut 06105 

SUMMERS, DR. WILLIAM C., Huxley College, Western Washington State Uni- 
versity, Bellingham, Washington 98225 

SUSSMAN, DR. MAURICE, Department of Biology, Brandeis University, Waltham, 
Massachusetts 02154 

SWANSON, DR. CARL PONTIUS, Department of Biology, The Johns Hopkins 
University, Baltimore, Maryland 21218 

SWOPE, MR. GERARD, JR., Blinn Road, Box 345, Croton-on-Hudson, New York 
10520 

SZABO, DR. GEORGE, Harvard School of Dental Medicine, 188 Longwood Avenue, 
Boston, Massachusetts 02115 

SZENT-GYORGYI, DR. ALBERT, Institute for Muscle Research, Marine Biological 
Laboratory, Woods Hole, Massachusetts 02543 

SZENT-GYORGYI, DR. ANDREW G., Department of Biology, Brandeis University, 
Waltham, Massachusetts 02154 

TANZER, DR. MARVIN L., Department of Biochemistry, University of Con- 
necticut, School of Medicine, Health Center, Hartford Plaza, Hartford, 
Connecticut 06105 

TASAKI, DR. ICHIJI, Laboratory of Neurobiology, National Institute of Mental 
Health, National Institutes of Health, Bethesda, Maryland 20014 

TAYLOR, DR. ROBERT E., Laboratory of Biophysics, National Institute of 
Neurological Diseases and Blindness, National Institutes of Health, Bethesda, 
Maryland 20014 



78 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

TAYLOR, DR. W. ROWLAND, Department of Oceanography, Chesapeake Ba\ 

Institute, The Johns Hopkins University, Baltimore, Maryland 21218 
TELFER, DR. WILLIAM H., Department of Biology, University of Pennsylvania, 

Philadelphia, Pennsylvania 19104 
DE TERRA, DR. NOEL, The Institute for Cancer Research, 7701 Burholme Avenue, 

Fox Chase, Philadelphia, Pennsylvania 19111 
TE\VINKEL, DR. Lois E., Department of Zoology, Smith College, Northampton, 

Massachusetts 01060 
THALER, DR. M. MICHAEL, University of California, San Francisco, California 

94102 

TRACER, DR. WILLIAM, The Rockefeller University, New York, New York 10021 
TRAVIS, DR. D. M., Department of Pharmacology, University of Florida, Gaines- 
ville, Florida 32601 
TRAVIS, DR. DOROTHY FRANCES, 1918 Northern Parkway, Greenberry Woods, 

Baltimore, Maryland 21210 
TRINKAUS, DR. J. PHILIP, Department of Biology, Yale University, New Haven, 

Connecticut 06520 

TROLL, DR. WALTER, Department of Environmental Medicine, New York Univer- 
sity, College of .Medicine, New York, New York 10016 
TWEEDELL, DR. KEN YON S., Department of Biology, University of Notre Dame, 

Notre Dame, Indiana 46556 
URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago, 

Chicago, Illinois 60637 
YALIELA, DR. IVAN, Resident Ecologist, Systematics-Ecology Program, Marine 

Biological Laboratory, Woods Hole, Massachusetts 02543 
YAM HOLDE, DR. KENSAL EDWARD, Oregon State University, Department of 

Biochemistry and Biophysics, Corvallis, Oregon 97331 
VILLEE, DR. CLAUDE A., Department of Biochemistry, Harvard Medical School, 

Boston, Massachusetts 02115 
VINCENT, DR. WALTER S., Department of Biology, University of Delaware, 

Newark, Delaware 19711 
WAINIO, DK. W. W., Bureau of Biological Research, Rutgers University, New 

Brunswick, New Jersey 08903 
WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge, 

Massachusetts 02138 
WALD, DR. RUTH HUBBARD, Biological Laboratories, Harvard University, 

Cambridge, Massachusetts 02139 
WALLACE, DR. ROBIN A., P. (). Box Y, Oak Ridge National Laboratory, Oak 

Ridge, Tennessee 37890 
WARNER, DR. ROBERT C., Department of Chemistry, New York University 

College of Medicine, New York, New York 10016 
WARREN, DR. LEONARD, Department of Therapeutic Research, University of 

Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 
WATERMAN, DR. T. H., 610 Klein Biology Tower, Yale University, New Haven, 

Connecticut 06520 

WATKINS, DR. DUDLEY TAYLOR, Department of Anatomy, University of Con- 
necticut, Farmington, Connecticut 06268 
WATSON, DR. STANLEY WAYNE, Woods Hole Oceanographic Institution, Woods 

Hole, Massachusetts 02543 



REPORT OF THE DIRECTOR 79 

WEBB, DR. H. MARGUERITE, Department of Biological Sciences, Goucher College, 
Towson, Maryland 21204 

WEBER, DR. ANNEMARIE, Department of Biochemistry, St. Louis University, 
St. Louis, Missouri 63108 

WEIDNER, DR. EARL, Rockefeller University, 66th Street and York Avenue, 
New York, New York 10021 

WEISS, DR. LEON P., Department of Anatomy, The Johns Hopkins University, 
School of Medicine, Baltimore, Maryland 21205 

WEISS, DR. PAUL A., The Rockefeller University, New York, New York 10021 

WEISSMANN, DR. GERALD, Professor of Medicine, New York LIniversity, 550 
First Avenue, New York, New York 10016 

WERMAN, DR. ROBERT, Department of Zoology, Hebrew University, Jerusalem, 
Israel 

WHITAKER, DR. DOUGLAS M., 3300 Hillcrest Drive, Apt. 209, San Antonio, 
Texas 78201 

WHITE, DR. E. GRACE, 1312 Edgar Avenue, Chamberslnirg, Pennsylvania 17201 

WHITING, DR. ANNA R., 535 West Yanderbilt Drive, Oak Ridge, Tennessee 37830 

WHITING, DR. PHINEAS, 535 Yanderbilt Drive, Oak Ridge, Tennessee 37830 

WHITTAKER, DR. j. RICHARD, Wister Institute of Anatomy and Biology, 36th 
Street at Spruce, Philadelphia, Pennsylvania 19104 

WICHTERMAN, DR. RALPH, Department of Biology, Temple University, Phila- 
delphia, Pennsylvania 19122 

WIERCINSKI, DR. FLOYD J., Department of Biology, Northeastern Illinois State 
University, 5500 North St. Louis Avenue, Chicago, Illinois 60625 

WIGLEY, DR. ROLAND L., National Marine Fisheries Service, Woods Hole, 
Massachusetts 02543 

WILBER, DR. C. G., Chairman, Department of Zoology, Colorado State Univer- 
sity, Fort Collins, Colorado 80521 

WILCE, DR. ROBERT THAYER, Department of Botany, University of Massa- 
chusetts, Amherst, Massachusetts 01003 

WILSON, DR. DARCY B., Department of Pathology, University of Pennsylvania, 
School of Medicine, Philadelphia, Pennsylvania 19104 

WILSON, DR. EDWARD ()., Department of Zoology, Harvard University, Cam- 
bridge, Massachusetts 02138 

WILSON, DR. T. HASTINGS, Department of Physiology, Harvard Medical School, 
Boston, Massachusetts 02115 

WILSON, DR. WALTER L., Department of Biology, Oakland University, Rochester, 
Michigan 48063 

WINTERS, DR. ROBERT WAYNE, Department of Pediatrics, Columbia University, 
College of Physicians and Surgeons, New York, New York 10032 

WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry, 
Albert Einstein College of Medicine, New York, New York 10461 

WOELKERLING, DR. WILLIAM J., Department of Botany, University of Wisconsin, 
Madison, Wisconisn 53703 

WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton, 
Massachusetts 01060 

WYSE, DR. GORDON A., Department of Zoology, University of Massachusetts, 
Amherst, Massachusetts 01002 



80 



ANNUAL KKPOKT OK THK MARINE BIOLOGICAL LABORATORY 



WYTTENBACH, DR. CHARLES R., Department of Zoology, University of Kansas, 
Lawrence, Kansas 66044 

YNTEMA, DR. C. L., Department of Anatomy, State University of New York, 
Upstate Medical Center, Syracuse, New York 13210 

YOUNG, DR. DAVID KENNETH, Bard College, Annandale-on-the-Hudson, New 
York, New York 12504 

ZACK, DR. SUMNER IRWIN, The Pennsylvania Hospital, University of Pennsyl- 
vania School of Medicine, Philadelphia, Pennsylvania 19104 

ZIGMAN, DR. SEYMOUR, University of Rochester School of Medicine and Den- 
tistry, 260 Crittenden Boulevard, Rochester, New York 14620 

ZIMMERMAN, DR. A. M., Department of Zoology, University of Toronto, Toronto 
5, Ontario, Canada 

ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, 
Kingston, Rhode Island 02881 

ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie, 
New York 12601 

ZULLO, DR. VICTOR A., Department of Geology, California Academy of Sciences, 
Golden Gate Park, San Francisco, California 94118 

Z\VEIFACH, DR. BENJAMIN, % AMES, University of California, San Diego, La 
Jolla, California 92073 



ASSOCIATE M EM BERS 



ABELSON, DR. AND MRS. PHILIP H. 

ACKROYD, DR. AND MRS. FREDERICK 

w. 

ADELBERG, DR. AND MRS. EDWARD A. 
ADELMAN, DR. AND MRS. WILLIAM J. 
ALLEN, Miss. CAMILLA K. 
ALTON, MRS. BENJAMIN (ELIZABETH 

MOEN) 

ANDERSON, DR. AND MRS. EVERETT 
ANGUS, DR. AND MRS. RALPH G. 
ANTHONY, MR. AND MRS. RICHARD A. 
ARMSTRONG, MRS. PHILIP B. 
ARNOLD, DR. AND MRS. JOHN 
BATON, DR. CATHERINE L. 
BACON, MR. ROBERT 
BACON, MRS. KATHERINE J. 
BAKALAR, MR. AND MRS. DAVID 
BALL, DR. AND MRS. ERIC G. 
BALI \NIIM :, DR. AND MRS. II. T., JR. 
BANKS, MR. AND MRS. W. L. 
BARBOUR, MRS. Lucius H. (ELIZABETH 

B.) 
BARROWS, MRS. ALBERT W. (MARY 

PRENTICE) 
B \RTO\V, MR. AND MRS. CLARENCE \Y. 



BARTOW, MRS. FRANCIS D. (S. R.) 
BEALE, MR. AND MRS. E. F. 
BERNHEIMER, DR. ALAN W. 
BIDDLE, DR. VIRGINIA 
BIGELOW, MRS. ROBERT P. (CAROLINE 

CHASE) 

BOETTIGER, DR. AND MRS. EDWARD G. 
BRADLEY, DR. CHARLES C. 
BRONSON, MR. AND MRS. SAMUEL C. 
BROWN, DR. AND MRS. DUGALD E. S. 
BROWN, DR. AND MRS. F. A., JR. 
BROWN, DR. AND MRS. THORNTON 

(SARAH MEIGS) 
BUCK, MRS. JOHN B. 

BUFFINGTON, MRS. ALICE H. 
BUEEINGTON, MRS. GEORGE (SARAH L.) 

BURDICK, DR. C. LALOR 

BURT, MR. AND MRS. CHARLES E. 

(KELEK FOUNDATION) 
BUSSER, DR. AND MRS. JOHN H. 
BUTLER, DR. AND MRS. E. G. 
CALKINS, MR. AND MRS. G. N., JR. 
CAMPBELL, MR. AND MRS. WORTHING- 

TON, JR. 
CARY, Miss CORNELIA L. 



REPORT OF THE DIRECTOR 



81 



CARLTON, MR. AND MRS. WINSLOW G. 
CARPENTER, MR. DONALD F. 
CASHMAN, MR. AND MRS. EUGENE R. 
CHAMBERS, DR. AND MRS. EDWARD L. 
CHENEY DR. AND MRS. RALPH H. 
CLAFF, DR. C. LLOYD 
CLARK, DR. AND MRS. ARNOLD M. 
CLARK, MR. AND MRS. HAYS 
CLARK, MRS. JAMES McC. (CYNTHIA) 
CLARK, DR. AND MRS. LEONARD B. 
CLARK, MRS. LEROY (ADNA A.) 
CLARK, MR. AND MRS. W. VAN ALAN 
CLEMENT, DR. AND MRS. A. C. 
CLEMENTS, MR. AND MRS. .DAVID T. 

COCHRAN, MR. AND MRS. F. MORRIS 

COFFIN, MR. AND MRS. JOHN B. 

COPELAND, DR. AND MRS. D. EUGENE 

CLOWES, MR. ALLEN W. 
CLOWES, DR. AND MRS. G. H. A., JR. 
(MARGARET J.) 

CONNELL, MR. AND MRS. W. J. 

COSTELLO, MRS. DONALD P. 

CRAMER, MR. AND MRS. IAN D. W. 

CRANE, MR. AND MRS. LOREN O. 

CRANE, MR. JOHN 

CRANE, JOSEPHINE, FOUNDATION 

CRANE, Miss LOUISE 

CRANE, MR. STEPHEN 

CRANE, MRS. W. CAREY 

CRANE, MRS. W. MURRAY 

CROCKER, MR. AND MRS. PETER J. 

CROSSLEY, MR. AND MRS. ARCHIBALD 

M. 

CROWELL, MR. AND MRS. PRINCE S. 
CURTIS, DR. AND MRS. W. D. 
DAIGNAULT, MR. AND MRS. A. T. 
DANIELS, MR. AND MRS. BRUCE G. 
DANIELS, MRS. F. HAROLD 
DAY, MR. AND MRS. POMEROY 
DRAPER, MRS. MARY C. 
DuBois, DR. AND MRS. A. B. 
DuPoNT, MR. A. FELIX, JR. 
DYER, MR. AND MRS. ARNOLD 
EASTMAN, MR. AND MRS. CHARLES E. 
EBERT, DR. AND MRS. JAMES D. 
EGLOFF, DR. AND MRS. F. R. L. 
ELLIOTT, MRS. ALFRED 
ELSMITH, MRS. DOROTHY O. 



EWING, DR. AND MRS. GIFFORD C. 
FACHON, MRS. EVANGELINE M. 
FAXON, DR. NATHANIEL W. 
FENNO, MRS. EDWARD N. 
FERGUSON, DR. AND MRS. J. J., JR. 
FINE, DR. AND MRS. JACOB 
FIRESTONE, MR. AND MRS. EDWIN 
FISHER, MR. FREDERICKS., Ill 
FISHER, MRS. B. C. (ELLEN D. B.) 
FRANCIS, MR. AND MRS. LEWIS W., JR. 
FRIES, MR. AND MRS. E. F. B. 
FYE, DR. AND MRS. PAUL M. 
GABRIEL, DR. AND MRS. MORDECAI L. 
GAISER, DR. AND MRS. DAVID W. 

(MARY JEWITT) 

GALTSOFF, DR. AND MRS. PAUL S. 
GAMBLE, MR. AND MRS. RICHARD B. 
GARFIELD, Miss ELEANOR 
GAYTON, MR. GARDNER F. 
GELLIS, DR. AND MRS. SYDNEY 
GERMAN, DR. AND MRS. JAMES L., Ill 
GIFFORD, MR. AND MRS. JOHN A. 
GIFFORD, MRS. MAUDE VESTERGARD 
GIFFORD, DR. AND MRS. PROSSER 
( .IKFORD, MRS. W. M. 
GILBERT, DR. AND MRS. DANIEL L. 

GlLCHRIST, MR. AND MRS. JOHN M. 

GILDEA, DR. MARGARET C. L. 
GILLETTE, MR. AND MRS. ROBERT S. 
GOLDSTEIN, MRS. MOISE H., JR. 
GLAZEBROOK, MRS. JAMES R. 
GLUSMAN, DR. AND MRS. MURRAY 
GOLDMAN, DR. AND MRS. ALLEN S. 
GOLDRING, DR. IRENE P. 
GOOD, Miss CHRISTINA 
GRAHAM, DR. AND MRS. HERBERT W. 
GRANT, DR. AND MRS. THEODORE J. 
GRASSLE, MR. AND MRS. J. K. 
GREENE, MR. AND MRS. WILLIAM C. 
GREEN, Miss GLADYS M. 
GREIF, DR. ROGER L. 
GREER, MR. AND MRS. W. H., JR. 
GRUSON, MR. AND MRS. EDWARD 
GULESIAN, MR. AND MRS. PAUL J. 

(MINNIE H.) 

GUNNING, MR. AND MRS. ROBERT 
GUREWICH, DR. AND MRS. VLADIMIR 
HALLKTT, MR. AND MRS. DUDLEY W. 



82 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



HAMLEN, MRS. J. MONROE 
HANDLER, DR. AND MRS. PHILIP 
HANNA, MR. AND MRS. THOMAS C. 

(KATHERINE SHIPPEN) 
HARE, DR. AND MRS. II. GERALD 
HARRINGTON, MR. AND MRS. R. D. 
HARVEY DR. AND MRS. EDMUND N., JR. 
HARVERY, DR. AND MRS. RICHARD B. 

(JANET M.) 

HEFFRON, DR. RODERICK 
HILL, MRS. SAMUEL E. 

HlRSCHFELD, MRS. NATHAN B. 
HOCKER, MR. AND MRS. LON 

HOPKINS, MRS. HOYT S. 
HOUGH, MR. AND MRS. GEORGE A., JR. 
HOUGH, MR. AND MRS. JOHN T. 
HOUSTON, MR. AND MRS. HOWARD E. 

HUNZIKER, MR. AND MRS. HERBERT E. 
ISSOKSON, MR. AND MRS. ISRAKL 

JANNEY, AfR. AND MRS. WISTAR 
JEWETT, MR. AND MRS. G. P., JR. 
JOHNSON, MR. AND MRS. CRAWFORD 
JORDAN, DR. AND MRS. EDWIN P. 
KAHLER, MR. AND MRS. GEORGE A. 
KAHLER, MRS. ROBERT W. 
KAHN, DR. AND MRS. ERNEST 
KAIGHN, DR. AND MRS. MORRIS E. 
KEITH, MRS. HAROLD ('. 
KEITH, MR. AND MRS. JEAN R. 
KENNEDY, DR. AND MRS. EUGENE P. 
KENEFICK, MR. AND MRS. T. G. 
KEOSIAN, MRS. JESSIE 
KlNNARD, MR. AND MRS. L. R. 
KOHN, DR. AND MRS. HENRY I. 
KOLLER, DR. AND MRS. LEWIS R. 
LANCEFIELD, DR. AND MRS. DONALD 
LANGE, MRS. GEORGE M. 
LASSALLE, MRS. NORMAN 
LAWRENCE, MRS. MILFORD R. 
LAWRENCE, MRS. WILLIAM 
LAZAROW, DR. AND MRS. ARNOLD 
LEMANN, MRS. LUCY B. 
LENHER, DR. AND MRS. SAMUKI. 
LK\ INK, DR. AND MRS. KACIIMIKL 
Li'AV, DR. AND MRS. MILI<>\ 
LILLIK, MRS. KARI. ('. 
LOBB, PROF. AND MRS. JOHN 
LOEB, DR. AND MRS. ROBKRT P. 
LONG, MRS. G. C. 



LORAND, MRS. L. 

LOVELL, MR. AND MRS. HOLLIS R. 
LoWENGARD, MRS. JOSEPH 

LURIA, DR. AND MRS. S. E. 

MACKEY, MR. AND MRS. WILLIAM K. 

MAcNicHOL, DR. AND MRS. EDWARD J. 

MARSLAND, DR. AND MRS. DOUGLAS 

MARVIN, DR. DOROTHY H. 

MAST, MRS. S. O. 

MATHER, MR. AND MRS. FRANK J., Ill 

MAVOR, MRS. JAMES W., SR. 

MCCUSKER, MR. AND MRS. PAUL T. 

MCELROY, MRS. NELLA W. 

McGlLLICUDDY, DR. AND MRS. J. J. 
McKENZIE, MR. AND MRS. KENNETH 

C. 

McLANE, MRS. T. THORNE 

McLARDY, DR. AND MRS. TURN1-R 

VlEics, MR. AND MRS. ARTHUR 
MF.IGS, DR. AND MRS. J. WISTKR 
MICTZ, MRS. CHARLES B. 
MEYERS, MR. AND MRS. RICHARD 
MILKMAN, DR. AND MRS. ROGER D. 
MIXTER, MRS. W. J. 
MONTGOMERY, DR. AND MRS. CHARLES 

H. 

MOORE, DR. AND MRS. JOHN W. 
MORRELL, DR. FRANK 
MORSE, MR. AND MRS. CHARLES L., JR. 
MORSE, MR. AND MRS. RICHARD S. 
NEUBERGER, MRS. HARRY H. 
NEWTON, Miss HELEN K. 
NICHOLS, MRS. GEORGE QANE M.) 

NlCKERSON, MR. AND MRS. FRANK L. 

NORMAN, MR. ANDREW E. 

NORMAN, MR. AND MRS. ANDREW E. 

PACKARD, MRS. CHARLES 

PARK, MR. MALCOLM S. 

PARK, MR. AND MRS. FRANKLIN A. 

PATTEN, MRS. BRADLEY M. 

PENDERGAST, MRS. CLAUDIA 

PENDLETON, DR. MURRAY E. 

PENNINGTON, Miss ANNE H. 

PERKINS, MR. AND MRS. COURTLAND 

D. 

PERSON, DR. AND MRS. PHILIP 
PETERSON, MR. AND MRS. E. GUNNAR 
PHILIPPE, MR. AND MRS. PIERRE 
PORTER, DR. AND MRS. KEITH R. 



REPORT OF THE LIBRARIAN 



83 



PROSSER, MRS. C. LADD 
PUTNAM, MR. AND MRS. W. A., Ill 
RATCLIFFE, MR. THOMAS G., JR. 
RAYMOND, DR. AND MRS. SAMUEL 
REDFIELD, DR. AND MRS. ALFRED 
RENEK, MR. AND MRS. MORRIS 
REYNOLDS, DR. AND MRS. GEORGE 
REZNIKOFF, DR. AND MRS. PAUL 
RIGGS, MR. AND MRS. LAWRASON, III 
RIINA, MR. AND MRS. JOHN R. 
ROBERTSON, DR. AND MRS. C. \Y. 
ROBINSON, DR. AND MRS. DENIS M. 
ROGERS, MR. AND MRS. CHARLES E. 
Ross, MR. AND MRS. JOHN 
ROOT, DR. AND MRS. WALTER S. 
ROWE, MRS. WILLIAM S. 
RUGH, DR. AND MRS. ROBERTS 
RUSSELL, MR. AND MRS. HENRY D. 
RYDER, MR. AND MRS. FRANCIS D. 
SAUNDERS, DR. AND MRS. JOHN W. 
SAUNDERS, MRS. LAWRENCE 
SAVERY, MR. ROGER 
SCHLESINGER, MRS. R. WALTER 
SCHROEDER, MR. RlCHARD F. 
SEARS, MR. AND MRS. HAROLD B. 
SHEPRO, DR. AND MRS. DAVID 
SHEMIN, DR. AND MRS. DAVID 
SMITH, DR. FREDERICK 
SMITH, AIRS. HOMER P. 
SPEIDEL, DR. AND MRS. CARL C. 
STEINBACH, DR. AND MRS. H. B. 
STETTEN, DR. AND MRS. DEWrn, JR. 
STONE, MR. AND MRS. LEO 
STRATTON, MR. AND MRS. WINSTON 
STUNKARD, DR. HORACE 
STURTEVANT, MRS. P. 

SWANSON, DR. AND MRS. CARL P. 

SWEENY, DR. AND MRS. THOMAS D. 
SWOPE, MR. AND MRS. GERARD L. 
SWOPE, MR. AND MRS. GERARD, JR. 



SWOPE, Miss HENRIETTA H. 
TAYLOR, DR. AND MRS. W. RANDOLPH 
THOMAS, MR. AND MRS. LEWIS 
TODD, MR. AND MRS. GORDON F. 

TOLKAN, MR. AND MRS. NORMAN N. 

TOMPKINS, MR. AND MRS. B. A. 
TRACER, MRS. WILLIAM 
TURNER, MRS. ROBERT 
VALOIS, MR. AND MRS. JOHN 
WAKSMAN, DR. AND MRS. BYRON FI. 
WAKSMAN, DR. AND MRS. SELMAN A. 
WALLACE, DR. AND MRS. STANLEY L. 
WANG, DR. AND MRS. AN 
WARE, MR. AND MRS. J. LINDSAY 
WARREN, DR. AND MRS. SHIELDS 
WATT, MR. AND MRS. JOHN B. 
WEISBERG, MR. AND MRS. ALFRED M. 
WEXLER, MR. AND MRS. ROBERT 11. 
WHEATLEY, DR. MARJORIE A. 
WHEELER, MR. AND MRS. HENRY 
WHEELER, DR. AND MRS. PAUL S. 
WHEELER, DR. AND MRS. RALPH E. 
WHITELEY, MR. AND MRS. G. C., JR. 
WHITING, DR. AND MRS. PHINEAS W. 
WHITNEY, MR. AND MRS. GEOFFREY 
G., JR. 

WlCKERSHAM, MR. AND MRS. A. A. 

TlLNEY 

WlCHTERMAN, DR. AND MRS. RALPH 
WlLBER, DR. AND MRS. CHARLES G. 

WILHELM, DR. HAZEL S. 
WILSON, MRS. EDMUND B. 
WlTMER, DR. AND MRS. ENOS E. 
WOLFE, DR. CHARLES 
WOLFINSOHN, MR. AND MRS. WOLFE 
WRINCH, DR. DOROTHY 
WRINCH, DR. PAMELA N. 
YNTEMA, DR. AND MRS. CHESTER L. 
ZWILLING, MRS. EDGAR 



Y. REPORT OF THE LIBRARIAN 

The MBL Associates' gift for 1971 was $10,507 and the Library had the good 
fortune to be the sole recipient of their gift. This will make a tremendous differ- 
ence in our "book" section, as \ve have been unable to add to this area due to 
cutbacks in the budget. A number of committees were formed in 1970 for the 
purpose of recommending books that were essential to the MBL and many lists 



84 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

were prepared by the scientists. The Associates' gift will cover the cost of all 
the recommended books. 

In December we moved the reprints down to the basement stack where there 
is no room for expansion but the collection remains intact. What was the reprint 
floor now houses all journal titles from Proceedings of the National Academy of 
Sciences; India through to the end of the alphabet. The rest of the journal 
collection covers two and a half floors and there is now room for expansion for 
the next seven years. It took six of us (four from the Library and two from 
Buildings and Grounds) five weeks to move approximately 140,000 volumes. 
While we were making the major move we changed the arrangement of the titles 
so that the articles are no longer used in the title when arranged alphabetically. 
Journals are now easier to find, one does not have to remember if the title has the 
words "the," "de la," etc. 

The G. K. Hall Company published our catalog in 12 volumes and to date over 
70 sets have been sold. The MBL received a 10 per cent royalty fee after the 
first 35 sets have been sold. 

Xerox continues to be one of our major projects. We received over 4,400 
requests from other libraries during the year. There are now two xerox machines 
in the library throughout the year and during the summer months they run 
constantly. 

In 1971 we had 4,146 serial titles, 2,476 received currently. Our holdings now 
total 146,158 volumes. 



vi. RP:PORT OF THE TREASURER 

The market value of the General Endowment Eund and the Library Fund 
at December 31, 1971, amounted to $2,294,949 and the corresponding securities 
are entered in the books at a value of $1,555,338. This compares with values of 
$2,197,603 and $1,574,735, respectively, at the end of the preceding year. The 
average yield on the securities was 4.15% of the market value and 6.13% of 
the book value. Uninvested principal cash was $7,757. Classification of the 
securities held in the Endowment Fund appears in the Auditor's Summary of 
Investments. 

The market value of the Pooled Securities at December 31, 1971, amounted 
to $870,538 as compared to book values of $667,280. These figures compare 
with values of $770,487 and $662,428, respectively, at the close of the preceding 
year. The average yield on the securities was 3.53% of the market value and 
4.61% of the book value. Uninvested principal cash was in the amount of $2,295. 

The proportionate interest in the Pool Fund Account of the various funds, as 
of December 31, 1971, is as follows: 

Pension Funds 26.12% 

General Laboratory Investment. ... 19.83% 

F. R. Lillie Memorial Fund 2.16% 

Anonymous Gift -74% 



REPORT OF THE TREASURER 85 



Other : 



Bio Club Scholarship Fund -56% 

Rev. Arsenius Boyer Scholarship Fund .68% 

Gary N. Calkins Fund -65% 

Allen R. Alemhard Fund . .12% 

Lucretia Crocker Fund . . 2.35% 

E. G. Conklin Fund .40% 

Jevvett Memorial Fund. .20% 

M. H. Jacobs Scholarship Fund .28% 

Herbert W. Rand Fellowship 20.00% 

Mellon Foundation 9.44% 

Mary Rogick Fund . 2.07% 

Swope Foundation 5.20% 

Clowes Fund 9.20% 

Donations from MBL Associates for 1971 amounted to $10,507 as compared 
with $9,724 for 1970. Unrestricted gifts from foundations, societies and com- 
panies amounted to $32,947. 

During the year we administered the following grants and contracts: 

Investigators Training MBL Institutional 

5 NIH 4 NIH 1 NIH 

4 NSF 2 NSF 1 NSF 

1 ONR 1 Sloan 1 AEC 

1 WHIH 
1 EPA 
1 MWPC 

13 7 3 

The majority of federally funded grants and contracts provided for reimburse- 
ment of indirect costs on a cost per square foot basis, for the laboratory space 
assigned to a particular research project. A provisional rate of $10.00 per square 
foot is still in effect. The space cost basis is also applicable to NSF training grants, 
but actual funding was at a considerably lower level due to limited granting agency 
funds. Indirect costs for NIH training grants are computed at a rate of 8% of 
allowable direct costs. 

The following is a statement by the Auditors: 

To the Trustees of Marine Biological Laboratory, Woods Hole, Massachusetts: 

We have examined the balance sheet of Marine Biological Laboratory as 
of December 31, 1971, the related statements of operating expenditures and 
income and funds for the year then ended. Our examination was made in 
accordance with generally accepted auditing standards, and accordingly 
included such tests of the accounting records and such other auditing proce- 
dures as we considered necessary in the circumstances. We previously 
examined and have reported on 1970 financial statements. 



86 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

In our opinion, the aforementioned financial statements (pages 87 to 91) 
present fairly the financial position of Marine Biological Laboratory at 
December 31, 1971 and 1970 and the results of its operations for the years 
then ended and the changes in funds for the year ended December 31, 1971 
in conformity with the accounting principles referred to in Note A to the 
financial statements applied on a consistent basis. 

The supplementary schedules (pages 92 to 93) included in this report \vere 
obtained from the Laboratory's records in the course of our examination and, 
in our opinion, are fairly stated in all material respects in relation to the 
financial statements, taken as a whole. 

Boston, Massachusetts 

March 30, 1972 LYHRAXD, Ross BROS. AND MONTGOMERY 



REPORT OF THE TREASURER 87 



MARINE BIOLOGICAL LABORATORY 
BALANCE SHEET 

December 31, 1971 and 1970 

Investments 

1971 1970 

Investments held by Trustee: 

Securities, at cost (approximate market quotation, 1971 

$2,294,949; 1970 $2,173,414) $1,555,338 $1,573,362 

Cash.. 7,757 2,641 



1,563,095 1,576,003 

Investments of other endowment and unrestricted funds: 

Pooled investments, at cost (approximate market quotation, 

1971 $870,538; 1970 $770,487) less $5,728 temporary in- 
vestment of current fund cash 661,552 656,700 

Other investments. . 725,151 1,125,150 

Cash 2,295 485 

Due from current fund. . 94,412 65,176 



$ 3,046,505 $ 3,423,514 



Plant Assets 

I. and, buildings, library and equipment 12,443,510 9,662,611 

Less allowance for depreciation (Note A) 2,161,247 1,899,406 



10,282,263 7,763,205 

Construction in progress 2,476,261 

Investments at cost (approximate market quotation, 1971 

$537,115; 1970 $536,875) 737,881 712,745 



;i,020,144 $10,952,211 



Current Assets 

Cash., 199,877 276,166 

Temporary investment in pooled securities 5,728 5,728 

Accounts receivable (U. S. Government, 1971 $69,395; 1970- 

$52,621) 248,396 147,881 

Inventories of supplies and bulletins 44,999 41,749 

Other assets 7,798 10,310 

Due to endowment funds. . (94,412) (65,176) 



$ 412,386 $ 416,658 



88 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 

MARINE BIOLOGICAL LABORATORY 
BALANCE SHEET 

December 31, 1071 and 1970 

Invested Funds 

1>>71 
Endowment funds given in truM for benefit of the Marine Biological 

Laboratory.... $ 1,563,095 $ 1,576,003 

Endowment funds for awards and scholarships: 

Principal.. 427,702 427,702 

Unexpended income. . 55,591 51,458 

483,293 479,160 

Unrestricted funds functioning as endowment. 779,190 1,179,190 

Retirement fund. . 279,407 246,833 

Pooled investments accumulated loss (58,480) (57,672) 



8 3,046,505 $ 3,423,514 



1 'I nut Funds 

Funds expended lor plant, less retirements 12,443,510 1 1,797,632 

Less allowance for depreciation charged thereto 2,161,247 1,899,406 



10,282,263 9,898,226 

Accounts payable 341,240 

Unexpended plant funds. . 737,881 712,745 



.1,020,144 $10,952,211 



Current Liabilities and Funds 

Accounts payable and accrued expenses 31,485 10,309 

Advance subscriptions 34,338 34,275 

Unexpended grants research.. 47,837 66,324 

Unexpended balances of gifts for designated purposes 39,725 25,020 

Current fund. . 259,001 280,730 



412,386 $ 416,658 



The accompanying note; JN ,m integral part of the linancial statements. 

N >" 1 ' A. An minting Prinn f>les: The following accounting principles have been reflected in 
the accompanying financial statements: 

1. Investments are stated at cost. 

2. Investment income is recorded on a cash basis. 

3. Operating income is recorded when earned. 

4. Expenses are recorded on an accrual basis. 

5. Depreciation has been provided for plant assets at annual rates ranging from 1% to 5% of 
the original cost of the asset -,. 



REPORT OF THE TREASURER 



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90 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 





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REPORT OF THE TREASURER 91 

MARINE BIOLOGICAL LABORATORY 
STATEMENT OF FUNDS 

Year Ended December 31, 1971 

Balance Gifts and Invest- Used for Other Balance 

December Other ment Current Expcndi- December 

31, 1970 Receipts Income Expenses tures 31, 1971 

$ 37,054 
Invested funds $3,423,514 (400,000) (2) $155,420 $131,366 $38,117 $3,046,505 



Unexpended plant funds $ 712,745 309,911 24,792 309,567 $ 737,881 



Unexpended research 

grants... . $ 66,324 722,145 740,632 $ 47,837 



Unexpended gifts for 

designated purposes $ 25,020 25,242 10,507 30 $ 39,725 



(74,056) (1) 
Current fund. . .$ 280,730 400,000(2) 347,673 $ 259,001 



$1,020,296 $180,212 $882,505 $695,387 



Gifts and grants tor 

facilities 

construction 309,911 

Other gifts and receipts 25,242 

Grants for research, 

training and 

support. 722,145 

Appropriated from 

current income 

and other. 37,054 

(1) Excess of current 
expenditures over 

income (74,056) 

(2) Transfer from 
invested funds. . 



$1,020,296 

Expended for new 
laboratory and 
dormitory dining 
hall . . . 657,240 

Scholarship awards. . . 11,115 

Payments to pen- 
sioners 10,672 

Loss on sale of 

securities 16,330 

Other. 30 



$695,387 



The accompanying note A is an integral part of the financial statements. 



92 



ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 



MARINE BIOLOGICAL LABORATORY 



SUMMARY OF INVESTMENTS 



IVcL-niber 31, 1971 



Securities held by Trustee: 
General endowment fund: 
U S Government securities 


Cost 

$ '5,065 


Per- 
cent 

of 
Total 

2.0 


Market 

Quotations 

$ 26,070 


Per- 
cent 

of 
Total 

1.4 


Investment 
Income 
1071 

$ 1,803 


Corporate bonds 


640,163 


51.4 


565,180 


30.0 


36,203 


Preferred stocks 


84,770 


6.8 


61,318 


3.3 


3,385 


Common stocks . 


495,848 


39.8 


1,231,644 


65.3 


35,313 
















1,245,846 


100.0 


1,884,212 


100.0 


76,704 


General educational board 
endowment fund : 
U S Government securities 


51,112 


16 5 


53,183 


P 9 


3,698 


Other bonds 


146,965 


47 5 


118,592 


289 


9,590 


Preferred stocks 


15,476 


5 


7,024 


1 7 


579 


Common stocks 


95,939 


31.0 


231,938 


56 5 


4,763 
















309,492 


100.0 


410,737 


100.0 


18,630 


Total securities held by Trustee 


$1,555,338 




$2,294,949 




95,334 


Investments of other endowment and 
unrestricted funds: 
Pooled investments: 
U. S. Government securities. . . 


96,397 


14 4 


98 167 


11 ? 


7,194 


Corporate bonds 


89,200 


13 4 


73 686 


8 5 


5,299" 


Preferred stocks. . . 


60,158 


90 


56 550 


6 5 


3,145 


Common stocks 


421,525 


63.2 


642,135 


73 8 


15,095 
















667,280 


100.0 


$ 870,538 


100.0 


30,733 


Less temporary investment of 
current fund cash 


5*7 ~) Q 








249 
















661,552 








30,484 


< Mher investments: 
1 S. Government securities 


>7 938 








1,133 


Other bonds 


IS 0?9 








750 


Common stocks . 


49 635 








? 823 


Real estate 


17,549 










Short-term commercial notes 


615,000 








35,485 




725,151 








40,191 



REPORT OF THE TREASURER 93 

MARINE BIOLOGICAL LABORATORY 
SUMMARY OF INVESTMENTS CONTINUED 

December 31, 1971 

Investmen t 

Income 
Cost 1971 

Total investments of other 
endowment and 
unrestricted funds. . $1,386,703 70,675 



Total 166,009 

Custodian's fees charged thereto 10,589 



Investment income distributed to 

invested funds. 155,420 



Plant investments: 

Federal agency and corporate bonds. . . . 140,000 6,252 

Common stock.. . 595,010 18,436 

Preferred stock.. 2,871 104 



$ 737,881 24,792 



Current investments: 

Temporary investment in 

pooled securities $ 5,728 249 



Total investment income. $180,461 



Reference: Biol. Bull, 143: 94-111. (August, 1972) 



THE SKXSORY BASIS OF HOST RECOGNITION BY 
SYMBIOTIC SHRIMPS. GEXUS BETAEUS 1 

BARRY W. ACHE AND DEMOREST DAVENPORT 

I)fpiirhiii~iit <>f l-tiolot/icul Sciences, University of California, 
Santa Barbara, California 93106 

Animals living in symbioses serve as excellent material for the analysis of ex- 
ternal stimuli controlling adaptive behavior. To a mobile partner, the host orga- 
nism represents a principal source of environmental stimuli, a source easily 
manipulated by the investigator. Experimental analyses of symbiotic relationships 
have demonstrated that chemical substances of host origin elicit host-oriented be- 
havior in crustacean species associated with pelecypods (Sastry and Menzel, 1962), 
polychaetes (Carton, 1 C >(>S: Davenport. Camougis and Hickok, 1960), echinoids 
(Gray, McClosky and \Yiehe. 19(>8). and amphinurans (Webster, 1968). These 
studies focused primarily on the role of chemical stimuli in effecting the respective 
symbiotic relationships. In surveying earlier work" on crustacean orientation, how- 
ever, Pardi and Papi (1961) note that even such relatively simple behavioral 
responses as kinetic and tactic orientation appear to be governed by higher neural 
centers, sometimes utilizing information from multiple sensory modalities. More 
recently, the interaction of multi-modal stimuli has been demonstrated to elicit 
and direct feeding behavior in several species of decapod crustaceans (Hiatt, 1948; 
Symons. 1964 ; Hazlett. 1968). 

The carideans Bctacus harjonli ( Kingsley ) and Bctacus inaci/initieae Hart are 
two of five species of betaeid shrimps adapted to a symbiotic existence (Hart, 
1964). Bctacus harjordi occurs in the mantle cavity of all eight species of Cali- 
fornia abalone. Haliotis spp. (Cox, 1962; Hart, 1964). B. inacc/initicac associates 
predominantly with the homochromous giant red sea urchin, Strongylocentrotus 
franciscanus (Agassiz) and occasionally with the purple urchin, S. f>nr[>itnitiis 
(Simpson) (Ache, 1970; Hart, 1964). Laboratory observations indicate that 
adult shrimps of both species leave their hosts during dark periods and return 
directly from distances up to 1 m away within a few minutes of the onset of light. 
(Ache, 1970). In doing so. they provide a behavioral response, i.e., tactic locomo- 
tion toward the host, suitable for analysis of the stimuli mediating distant host 
recognition. 

The present investigation attempts to elucidate and compare the sensory bases 
of the host location behavior of B. liarjordi and B. macginitieae and to explain the 
apparent specificity of their respective relationships in terms of the sensory compe- 
tence of the shrimps. 

3 Conducted under Contract NONR 4222(03) with the Office of Naval Research and 
' .rant \*o. NB04372 from the United States Public Health Service. 

2 Present addiw Department of Biological Sciences, Florida Atlantic University, Boca 
ton, Florida 33432. 

94 



HOST RECOGNITION BY SHRIMP 



95 



MATERIAL AND MKTIIODS 
( Organisms 

Specimens of Bctacns harfordi and B. Jinict/hiificae, 1.0-3.5 cm total length, 
were collected along with their respective hosts, the ahalones, H allot is corruc/ata 
Gray, H. rufesccns Swainson, and H. craclicrodii Leach and the urchin, Strongylo- 
centrotits franciscanits, from stibtidal populations in the Santa Barbara area. 
Shrimps were maintained in the laboratory isolated from their hosts on a diet of 
frozen Artonia. Shrimps were utilized for experimentation between the 2nd and 
10th days of holding. Hosts or other organisms to be tested as potential sources 
of stimuli (test organisms) were held without feeding and utilized within three 
days of laboratory confinement. 

Apparatus 

Two types of choice apparatus were utilized to quantify the host-oriented 
behavior of Betaeiis. One apparatus was simply a large (l.Ox 1.3 X 0.2 m) 
seawater-filled rectangular tank or arena. Seawater was continuously introduced 
via four inlet tubes, one located in each corner of the tank, and maintained at a 



o 







-S 




FIGURE 1. Diagram (top view) of two-celled choice apparatus: C test or control cells; 
I seawater inlets; O seawater outlets; S transparent plexiglass screen; R removable re- 
lease cylinder. Arrows indicate direction of flow into choice area. Inset is transverse view 
of continuous opening along bottom of plexiglass screen. Arrows indicate flow through this 
opening. 



96 BARRY W. ACHE AND DKMOREST DAVENPORT 

depth of 15 cm by two clear plastic standpipe drains centered on the longer axis 
of the tank. Plastic-coated screens fitted into each of the corners served to con- 
fine test organisms within approximately 15 cm of the seawater inlets, yet did not 
retard the movement of shrimps into or out of the corner compartments. A remov- 
able length of 14 cm diameter clear plastic tubing centered in the tank served as a 
release point for shrimps. The overhead fluorescent lamps of the room supplied 
relatively uniform illumination to the apparatus. 

Shrimp behavior was also quantified in a 2-celled choice apparatus designed 
to be compatible with the fast-moving Betaeus yet retain the binomial simplicity 
of a conventional Y maze. The apparatus (Fig. 1) incorporated a 33 X 4o X 12 cm 
opaque white polyethylene pan fitted with a T-shaped transparent plastic divider 
to form two small compartments (herein referred to as test and control cells) 
and a larger compartment (herein referred to as the choice area). A baffled 
opening in the transverse partition (inset, Fig. 1) allowed seawater introduced 
into the test and control cells to flow into the choice area w r here it was removed 
by two constant-level siphons. An input of 7.0 ml/sec of new seawater to each 
cell produced an even, laminar flow of approximately 5 mm/sec along the bottom 
of the choice area (arrows, Fig. 1 ). A removable opaque cylinder allowed intro- 
duction of single shrimp into the choice area with minimal directional bias. A 7.5w 
frosted incandescent lamp centered over the apparatus 50 cm from the water's sur- 
face supplied even, low-intensity illumination to the choice area. The 2-celled 
choice apparatus was adapted for detailed analysis of visual stimuli by replacing 
the open-bottomed transparent partition with a watertight transparent partition to 
insure chemical isolation of all three compartments and by removing the seawater 
inlet and outlet tubes to create a static system. A 4 cm strip of opaque white 
plastic attached to the transparent panel to increase the separation between the 
two cells enhanced the resolution of right and left choices. 

Procedure and data analysis 

Shrimps were isolated from their hosts up to 10 days prior to testing. Symons 
(1964) has noted that the ability of either chemical or tactile stimuli to evoke feed- 
ing in the crab Hemigrapsus oregonensis increases over a 10 day period of starva- 
tion, approaching the ability of combined tactile-chemical stimuli to elicit the same 
response by day 10. To minimize any bias due to threshold change, all our 
experiments w r ithin a series were performed in as short a time as possible. 

Arena apparatus Ten minutes prior to introduction of the shrimps two to 
four host organisms were placed in each of two diagonally opposite corner com- 
partments. As controls, three non-host organisms, the seastar Dermasterias 
iinbricata, were placed in each of the two remaining corner compartments. The 
presence of other live organisms in the control compartments minimized the 
possibility of the data reflecting a generalized response to any animate object. 
The animal complement of each compartment was equated by weight ( 50 g) to 
the mean weight of three seastars, 320 g. Twelve or 15 shrimp selected from 
a group of 120-125 individuals were dip-netted into the central release cylinder, 
held for 5 minutes, and then released by removing the cylinder. The location of 
each individual was recorded 30 minutes following release. This sequence was 
ied eight times for any one set of test conditions, with the contents of the 



HOST RECOGNITION BY SHRIMI' 

corner compartments moved clockwise one compartment with each repetition. 
Mucus and debris were wiped from each compartment with each rotation. 

The hypothesis that the terminal distribution of shrimp was randomly divided 
between the two pair of corner compartments was tested by comparing the total 
number of shrimps found within the two host-containing compartments with the 
number found within the two control compartments. In each experiment, the 
probability of obtaining the observed distribution was tested for its association 
with a theoretical distribution of 0.50-0.50, utilizing chi-square. 

Tza'o-celled choice apparatus. Experimental protocol with this apparatus 
consisted of introducing potential sources of stimuli to one or both of the cells, 
allowing 10 minutes for equilibration, and monitoring the time (t <S 6 min) re- 
quired for each of 30 shrimps, individually and sequentially introduced, to leave 
the release point, traverse the length of the choice area, and contact the transparent 
partition delimiting one of the two cells. Thirty shrimps, selected at random from 
groups of 70-80 individuals, were utilized in each experiment. Experiments 
comprising an experimental series utilized the same group of 70-80 shrimps. A 
new group of 70-80 shrimps was obtained for each experimental series. The 
contents of the two cells were exchanged in each experiment after testing one-half 
of the M) shrimps, the exchange being accompanied by washing and refilling of 
the apparatus with fresh seawater. Data displaying a significant non-experimental 
bias (P < 0.05) to either cell were voided and the experiment repeated. This 
practice, which required repetition of approximately 8% of the experiments, effec- 
tively controlled for transient bias (e.g., obstruction of an inlet tube by participate 
material) that may have occurred during the course of an experiment. 

The number of shrimps reaching criterion, i.e., those contacting the trans- 
parent partition delimiting the host-containing cell, was compared by chi-square 
analysis to an expected distribution in which 50 r /r of the total number of indi- 
viduals making a choice go to each cell. This value is subsequently referred to as 
"x~ choice." In many experiments, a number of individuals failed to move, or 
move but failed to reach criterion within the 6 minute experimental period. For 
an experiment questioning the relative attractiveness of a stimulus situation, the 
number of organisms not reaching criterion represents significant information. 
Formulation of this category involves combining the number of individuals not 
moving, those moving but not reaching criterion, and those choosing the control cell. 
Although detailed a priori knowledge of how shrimps making a choice relate to 
those not stimulated to move remains unknown, individuals of all three combined 
groups can be considered as not displaying a positive response to the stimulus 
source of a particular experiment. This rationale is not without precedent (see 
Davenport, 1950). Differences in the numbers of shrimps failing to reach criterion 
for any two experiments of a series were tested for significance with a two-way 
contingency analysis adjusted for continuity. (Simpson, Roe, and Lewontin, 1960, 
page 190)! 

RESULTS 
Tlie Betaeus-Haliotis association 

Experiments utilizing arena apparatus. These experiments attend to the ques- 
tion : Do shrimps collected from the host, H. rnfescens, require both chemical and 



98 



BARRY W. ACHE AND DKMOREST DAVENPORT 



TABLE I 
Arena experiments: B. harfordi-H. rufescens vs. D. imbricata 









Shrimps choosing 






Experiment 


Stimulus modality 
removed 


Shrimps 
tested 




X 2 

choice 


P 












Host 


Control 






1 


None 


96 


82 


8 


72.3 


< 0.005 


2 


Chemical 


111 


29 


38 


0.12 


0.50-0.75 


3 


Visual 


11 1 


78 


25 


27.2 


<0.005 


4 


None, hosts in .ill conu-o 


96 


34 


48 


2.38 


0.10-0.25 



visual stimuli of host origin to effect host location? Shrimps were permitted to 
choose between two corner compartments containing host abalone and two con- 
taining the control organism while visual or chemical stimuli were selectively 
removed from the choice situation. Table I summarizes the results of these 
experiments which extended over an eight day period. With both visual and 
chemical stimuli present, shrimps preferentially selected those compartments con- 
taining abalone, H. rnfcsccns, over those containing the seastars (Experiment 1). 
With all organisms contained in clear glass 4 liter beakers to eliminate chemical 
stimuli from the choice situation, but otherwise identical protocol maintained, the 
differential response elicited in Experiment 1 was abolished (Experiment 2). 
However, with the corner compartments covered with eight layers of white 
cheesecloth so as to exclude visual stimuli from the choice situation yet not alter 
the flow characteristics of the system, shrimps exhibited preferential selection of 
the abalone-containing compartments (Experiment 3). To further eliminate the 
possibility that bias existed in the experimental procedure itself, host abalones 
were substituted for seastars in the control compartments, i.e., all compartments 
contained host abalone, and Experiment 1 was repeated (Experiment 4). No sig- 
nificant difference occurred in the number of shrimps selecting either of the two 
pair of host-containing compartments. 

Testing shrimps in groups introduced the possibility of bias due to shrimp- 
shrimp interactions. The 30 minute experimental period allowed multiple re- 
sponses by any one shrimp, which were observed to occur in a small percentage 
of the trials. For these reasons, and since more than 50% of the shrimps entered 
a corner compartment within the first 3-4 minutes of the 30 minute experimental 
period, further analysis utilized the 2-celled choice appartus, designed for short 
term observation of individual organisms. 

Experiments utilizing 2-ccllcd choice apparatus. The question considered in 
the preceding group of experiments was again tested as a basis for comparison of 
the two techniques. In these experiments, shrimps collected from the abalone, 
H. cornigata, were permitted to choose between : Experiment 1 a test cell con- 
taining seawater only, Experiment 2 as Experiment 1 only with the transverse 
partition covered with an opaque screen to remove visual stimuli from the choice 
situation, Experiment 3 as Experiment 1 only with the abalone contained in a 
clear glass 4-liter beaker to remove chemical stimuli from the choice situation and 
Experiment 4 two control cells containing seawater only. Table II (Series 1) 
summarizes the results of these experiments. Significantly different choice be- 



HOST RECOGNITION BY SHRIMP 99 

tween test and control cells was elicited only in the presence of chemical stimuli 
of host origin. Essentially the same number of shrimps located the host-containing 
cell when only chemical stimuli were present (Experiment 2) as when both visual 
and chemical were present, e.g., the experimentally unaltered situation (Experi- 
ment 1). 

To gain a fuller understanding of the results of Series 1 experiments, it was 
necessary to know if the presence of chemical stimuli triggered a response to cur- 
rent, since chemical stimuli were always presented in association with a directional 
flow of water emanating from the test and control cells. Table II (Series 2) sum- 
marizes the results of experiments designed to answer this question. 

Shrimps in Series 2 experiments were collected from the host abalone, H. 
rufescens. Experiment 1 represents the experimentally unaltered choice situation. 
To determine if current alone had any effect, shrimps were permitted to discrimi- 
nate between a test cell containing the model abalone and a seawater control cell 
both with (Experiment 2) and without (Experiment 3) a current in the appa- 
ratus. To eliminate current, the seawater inlets were closed. Substituting a 
model abalone for a live one in the test cell allowed presentation of visual stimuli 
without chemical stimuli while retaining the directional flow. The model consisted 
of a paraffin-filled abalone shell with 1.5 cm wide "epipodium" of black tape ex- 
posed beneath the ventral edge of the shell. As live abalone remained stationary 
when placed in the apparatus, a static model was judged an acceptable substitute. 
As can be seen, differential choice between test and control cells was not elicited 
in either experiment. Likewise, a test of association comparing the number of 
shrimps choosing the model-containing cell vs. the number not choosing it between 
the two experiments indicates no difference in the response of the shrimps x 2 
0.223, P -0.50-0.75). It appears that current itself does not effect the activity 
of the shrimps nor their response to visual stimuli. 

To determine if the presence of non-directional chemical stimuli had any effect, 
shrimps were permitted to discriminate between test and control cells when non- 
directional chemical stimuli of host origin were present throughout the system, 
but in the absence of a flow (Experiment 4). Two specimens of H. rujescens 
(350 g), confined in a perforated plastic cup and swirled in the choice area of 
the apparatus for 1 minute prior to introduction of each shrimp, served to intro- 
duce non-directional chemical stimuli into the system. Assuming host effluents 
had an effective time stability of at least 6.0 minutes, host effluent was present in 
the choice area throughout the maximum time interval allowed for choice. This 
assumption, of course, could only be confirmed by a positive result, i.e., by obtain- 
ing a significant change in response on the addition of such non-directional chemi- 
cal stimuli. Differential choice was elicited in favor of the model-containing cell, 
suggesting that the presence of non-directional chemical stimuli may enhance the 
stimulus value of visual cues characterizing the model host. A test of association 
comparing the number of shrimps choosing the model-containing cell vs. the 
number not choosing it in this (Experiment 4) and in the control situation (Ex- 
periment 3- no current, no chemical ) , however, indicates that no significant in- 
crease in the level of activity occurred in the presence of the non-directional 
chemical stimuli (x~ -0.178, P 0.50-0.75). It appears that non-directional 



100 



BARRY W. ACHE AND DEMOREST DAVENPORT 



chemical stimuli alone are not sufficient to affect the activity of the shrimps, al- 
though they may enhance directed activity in the presence of visual cues. 

To determine if non-directional chemical stimuli serve to trigger a response to 
current, shrimps were permitted to discriminate between test and control cells 
when non-directional chemical stimuli of host origin were presented simultaneously 
with a directional flow (Experiment 3). No differential choice was elicited be- 
tween the test and control cells. However, the method of introducing the chemical 
stimuli in this experiment should have dispersed host effluent throughout all com- 
partments of the apparatus. Since a current was flowing under the transparent 
partition from both test and control cells, no difference should have existed in the 
stimulus pattern characterizing the two cells except for the visual stimuli of the 
model-containing cell. Experiments 2 and 5 indicate that visual stimuli with or 
without current elicit little activity. Thus, the combined number of shrimps 
reaching either cell can be considered as being most characteristic of the response 
to this stimulus situation. A test of association comparing the total number of 
shrimps choosing either cell t's. the number not choosing either cell in Experiment 
5 and in the basic host response (Experiment 1 visual, current, and directed 
chemical stimuli of host origin I . indicates no significant difference in the level of 
activity (P > 0.995). Host-oriented locomotion in the shrimp B. harfordi appears 



TABU: 1 1 
Two-celled ihoice experiments: B. harfordi 



Experiment 


Content^ of 
test cell 


Stimulus 
modalitir- 
present* 


Shrimps choosing 


X 2 
Choice 


P 


IV-t 


Control 


Series 1 














1 


H. corrngata 


V, DC 


26 


1 


23.0 


< 0.005 


2 


H. corrugatu 


DC 


25 


2 


19.6 


<( J.005 


3 


H. corrugata 


V 


14 


6 


1.60 


0.10-0.25 - 


4 


Sea water 





4 


4 








Scries 2 














1 


H. rufescens 


V, DC, C 


26 





26.0 


< 0.005 


2 


H. rufescens 


V, C 


2 


2 








3 


H. rufescens 


V 


4 


3 


0.14 


0.50-0.75 


4 


H. ryfesiens 


V, NDC 


8 





8.00 


<0.005 


5 


H. rufescens 


V, NDC, C 


14 


10 


0.67 


0.25-0.50 


Series 3 














1 


H. crachcrodu 


DC 


25 





25.0 


< 0.005 


1 


Seawater 





5 


2 


1.29 


0.25-0.50 


3 


H. rufescens 


DC 


24 





24.0 


< 0.005 


4 


H. rormgata 


DC 


28 





28.0 


< 0.005 


5 


K. kelletia 


DC 


8 


8 








6 


V. franciscanus 


DC 


9 


4 


1.92 


0.10-0.25 


7 


M . * rt'uidata 


DC 


6 


8 


0.28 


0.50-0.75 


8 


, <lU[)tt 


DC 


7 


2 


2.78 


0.05-0.10 


9 


H . crachenidii 


DC 


27 


1 


24.2 


< 0.005 


10 


Seawater 





4 


7 


0.81 


0.25-0.50 



V, visual; DC, directed chemical; NDC, non-directed chemical; C, current. 



HOST RECOGNITION BY SHRIMP 



101 



to result from the ability of chemical stimuli to release a response to directional 
water currents in these shrimp. 

It was then asked: Is the distribution of the active substance (s) sufficiently 
restricted to explain the apparent specificity of association of the shrimps to 
molluscs of the genus Haliotis? Table II (Series 3) summarizes experiments 
extending over five consecutive days that permitted specimens of B. Jwrfordi 
collected from the abalone, H. cracherodii, to discriminate between a test cell con- 
taining individuals of one of seven different species of test organisms and a control 
cell containing only seawater. An opaque white plastic screen placed over the trans- 
verse partition occluded visual stimuli from the choice situation. Test organism com- 
plements were equated to 350 50 g wet weight. Significantly different choice 
was elicited by effluents of test organisms of the genus Ha! tot is (Experiments 1, 
3, 4, 9). Differential choice was not elicited by effluents of two other gastropods, 
the neogastropod Kclettia kcllctii (Experiment 5) and the archeogastropod Mc</a- 
tluira crcnulata (Experiment 7). Similarly, differential choice was not elicited by 
effluents of the echiuroid Urcchis canpo (Experiment 8) nor of the echinoid 
Strongylocentrotus jranciscan/is ( Experiment 6). both reported to be hosts of con- 
generic Betaeus species (Hart, 1964). Agreement of initial and terminal replicates 
of the basic host response (Experiments 1,9) suggests the lack of response in the 
latter experiments was not the result of a temporal change in responsiveness of the 
shrimps. 

The total number of shrimps locating tests cells containing Haliotis spp. was 
greater than for the non haliotid species. A test of association on the results of 
the most and least extreme distributions obtained with Haliotis effluents (Experi- 
ments 3, 4) indicates no significant difference between the numbers of shrimps 
locating the test cell in these experiments ( x 2 - 0.935, P -- 0.25-0.50). A test of 
association on the results of the least extreme distribution obtained with a Haliotis 
effluent (Experiment 3) and the least extreme distribution obtained with non-host 
effluent (Experiment 6), however, indicated a significant difference between the 
number of shrimps locating the test cells ( x ~ -22.9, P- C 0.005). It follows 
that the remaining and more extreme distributions obtained to non-host effluents 
are also significantly different from the distribution obtained in Experiment 3. 

The Betaeus-Strongylocentrotus association 

Experiments utilizing arena apparatus. The question was first asked whether 
shrimps in association with the urchin S. franciscanus require both chemical and 

TABLE III 

Arena experiments: B. inacginitieae S. franciscanus vs. I), imbricata 









Shrimps choosing 






Kxperiment 


Stimulus modality 
removed 


Shrimps 
tested 




X 2 
choice 


P 












Host 


Control 






1 


None 


111 


89 


13 


56.6 


< 0.005 


2 


Chemical 


111 


63 


25 


15.4 


<0.005 


3 


Visual 


96 


63 


12 


35.4 


< 0.005 


4 


None, heists iu all corners 


99 


44 


47 


0.09 


0.75-0.90 



102 BARRY W. ACHE AND DEMOREST DAVENPORT 

visual stimuli of host origin to effect host location. Shrimps were permitted to 
make a choice when presented with two corner compartments containing host 
urchins and two confining the control organism. Visual or chemical stimuli 
were then selectively removed from the choice situation. Table III summarizes 
the results of these experiments which extended over a period of 8 days. Experi- 
ment 1 represents the basic host-location response of shrimps in the arena appara- 
tus when neither chemical nor visual stimuli were altered, i.e., the "natural" 
stimulus condition. A significantly greater number of shrimps selected the two 
host-containing cells. When both the test and control organisms were contained 
in clear glass 4-liter beakers to eliminate chemical stimuli from the choice situation 
and an otherwise identical experimental protocol maintained (Experiment 2), 
significantly more shrimps still selected the host-containing compartments. Like- 
wise, with the corner compartments covered with eight layers of white cheesecloth 
so as to exclude visual stimuli yet not alter the flow characteristics of the system 
and retain chemical stimuli (Experiment 3), significantly more shrimps selected the 
host-containing compartments. Experiment 4, in which host sea urchins were sub- 
stituted for the seastars in the control compartments (i.e., all compartments con- 
tained host urchins) suggests that final distributions of this series of experiments 
were not biased by the experimental procedure itself. 

Experiments utilizing 2-ccIlcd choice apparatus. For the reasons previously 
described, more detailed analyses utilized the 2-celled choice apparatus. Repeti- 
tion of the above described experiments provided a basis for comparison of 
the two techniques of behavioral quantification. Table IV (Series 1) summarizes 
the results of these experiments. Shrimps were permitted to select between a 
test cell containing the host urchin and a control cell containing seawater only. 
In the basic stimulus situation, where both chemical and visual stimuli were ex- 
perimentally unaltered, significantly more shrimps selected the host-containing 
cell (Experiment 1). With the host urchin contained in a clear glass 4-liter 
beaker placed in the test cell and a seawater filled beaker placed in the control 
cell, significantly more shrimps still selected the host-containing cell (Experi- 
ment 2). With an opaque white plastic screen attached to the transverse parti- 
tion thus masking visual stimuli from the choice situation while not interfering 
with chemical stimuli, significantly more shrimps again selected the host-containing 
cell (Experiment 3). Neither stimulus modality acting alone, however, elicited 
host location to the extent that both did when presented together. Very few 
shrimps made a choice in the absence of any stimuli of host origin (Experiment 4). 
These data are in agreement with those of the arena experiments indicating that 
either chemical or visual stimuli of host origin are sufficient to effect host loca- 
tion by these shrimps. 

The question was then asked to what extent can the response to chemical 
stimuli explain the apparent specificity of the B. macginitieae-Strongylocentrotus 
association. Screening the transparent plexiglass divider with a thin sheet of 
opaque white plastic arranged so as not to alter the flow characteristics of the 
apparatus effectively blocked visual communication between the cells and the 
choice area, while allowing free passage of chemical cues. The mean weight 
of the test organisms utilized in each experiment was 200 25 g. In a series of 
xperiments extending over five consecutive days, shrimps were permitted to 



HOST RECOGNITION BY SHRIMP 



103 



TABLE IV 
Two-celled choice experiments: B. macginitieae 



Experiment 


Contents of 
test cell 


Stimulus 
modalities 
present* 


Shrimps choosing 


X 2 
Choice 


P 


Test 


Control 


Series 1 














1 


A', franciscanus 


V, C 


28 


1 


25.0 


< 0.005 


? 


S. franciscanus 


V 


22 





22.0 


< 0.005 


3 


S. franciscanus 


C 


22 


1 


19.2 


< 0.005 


4 


Seawater 





2 


2 







Series 2 














1 


S. franciscanus 


C 


25 





25.0 


< 0.005 


2 


Seawater 





4 


2 


0.66 


0.25-0.50 


3 


S. purpuratus 


C 


20 





20.0 


< 0.005 


4 


L. anamesus 


C 


5 


1 


2.66 


0.10-0.25 


5 


.s. parmmensis 


C 


8 


6 


0.57 


0.25 0.50 


6 


D. imbricata 


C 


8 


6 


0.57 


0.25-0.50 


7 


H. rufescens 


C 


9 


6 


0.60 


0.25-0.50 


8 


U. caupo 


C 


8 


5 


0.69 


0.25-0.50 


9 


S. franciscanus 


C 


21 


1 


18.2 


<0.005 


10 


Seawater 





3 


5 


0.50 


0.50-0.75 



* V, visual; C, chemical; , neither visual or chemical. 

discriminate between a test cell containing one of seven different species of test 
organism and a control cell containing seawater only (Table IV, Series 2). Only 
effluent of tbe congeneric echinoids, v$\ franciscanus, the natural host (Experi- 
ments 1, 9), and 5\ purpuratus, (Experiment 3), elicited differential choice be- 
tween test and control cells. Differential choice was not elicited by effluents of 
the non-host echinoid, Lytcchinus anamcsns (Experiment 4), nor the non-echinoid 
echinoderms, Stichopus parvimensis (Experiment 5) and Dermasterias imbricata 
(Experiment 6). Likewise, effluents of the abalone, Haliotis rufescens (Experi- 
ment 7), and the echiurid worm, Urechls canpo, (Experiment 8) both reported 
hosts for congeneric Beta-ens species (Hart, 1964), failed to elicit differential 
choice. A test of association indicates that the final distribution elicited by 
effluents of the urchin. S. purpuratus (Experiment 3), does not differ significantly 
from the more extreme of the two distributions elicited by effluents of the natural 
host (Experiment 1) ( x 2 - 1.46. P - 0.1-0.25). That the distribution elicited by 
non-strongyloid effluents differs significantly from that obtained in Experiments 1, 
3, and 9, is indicated by a test of association of the least extreme distribution 
obtained with S. franciscanus effluent (Experiment 9) and the least extreme dis- 
tribution obtained with a non-host effluent, that of the abalone, H. rnfcsccns 
(Experiment 7) (x~ - 13.3, P -- < 0.005). Only a few individuals responded 
in the absence of any stimuli of host origin (Experiments 2, 10). Agreement of 
initial and final repetitions of the basic host response (Experiments 1, 9) support 
the hypothesis that no change occurred in the response level of the shrimps during 
the duration of the experimenal period. 

The question was then asked to what extent can the response to visual stimuli 
of host origin explain the apparent specificity of the B. macginitieae-Stronglyocen- 
trotits association. These experiments utilized the static modification of the 2-celled 



104 



BARRY W. ACHE AND DEMOREST DAVENPORT 



choice apparatus in which chemical stimuli and the carrier flow are absent from 
the choice situation. As a preliminary experiment, shrimps were permitted to 
select between a cell curtaining the host S. franciscanus and a cell containing one 
of five different test organisms, the host. J>\ franciscanus, the abalone, Haliotis 
rufesccns, the holothuroid, Sticliopits pun'iinctisis, the alternate host, Strongylo- 
ccntrohts pnrpitratns, and the giant keyhole limpet, Mcgathnra crcnitlata. These 
animals represent the predominant, nun -sessile, macrobenthic fauna of the Santa 
Barbara collection site. All test organisms were equated for displacement volume 
(+ 50 ml). Shrimps selected the host-containing cell in all cases except in the 
pairing of the limpet, M ci/titli/tni, and the host urchin. These experiments were 
not pursued further, however, due to the difficulty in equating such diverse orga- 
nisms for "unit" characteristics. 

As an alternative approach to gaining an understanding of the visual basis of 
the shrimp-urchin association, an effort was made to determine which com- 
ponent (s) of the total visual pattern characterizing S. franciscanus is (are) 
utilized by the shrimps to effect visually-mediated host recognition. These experi- 
ments quantified the ability of shrimps to visually distinguish between two simul- 
taneously presented objects (Cell A and Cell B). Any shrimp not moving away 
from the release point by 5.5 minutes was touched on the telson with a camel's hair 
brush, which served as sufficient stimulus to initiate movement to criterion in 



TABLE V 

B. macginitieac: analysis of stimulus parameters visually 
t harm tcrizhig Strongylocentrotus franciscanus 



Experi- 
ment 


Cell 
A 


Shrimps 
choosing 
A 


Shrimps 
chooMnu 
B 


Cell 
B 


x- 
choice 


P 


1 


Urchin (9.0j* 


23 


7 


Urchin (6.4) 


8.52 


< 0.005 




Urchin (6.4) 


21 


9 


Urchin (4.5) 


4.80 


0.025-0.05 


2 


Spineless urchin (8.0) 


18 


12 


Intact urchin (4.0) 


1.20 


0.25-0.50 


3 


Disk (10.0) 


14 


16 


Urchin (7.5) 


1.34 


0.75-0.90 




Disk (10.0) 


18 


12 


Urchin (5.0) 


1.20 


0.25-0.50 




Disk (10.0) 


28 


2 


Urchin (3.5) 


22.4 


< 0.005 


4 


Disk (9.0) 


27 


3 


Patterned disk (9.0; 


19.2 


< 0.005 


5 


Black disk, white 


24 


5 


White disk, black 


12.5 


< 0.005 




l>kgd. (3.8) 






bkgd. (3.8) 






6 


Disk (9.0) 


20 


9 


Sri-rated disk (9.0) 


4.16 


0.025-0.05 




Disk (9.0i 


17 


13 


Square (7.9)** 


0.53 


0.25-0.50 




Disk (9.0) 


7 


23 


Square (8.9) 


19.2 


<0.005 




Disk (6.0) 


15 


14 


Square (5.3) 


0.03 


0.75-0.90 




Disk (9.0) 


14 


15 


Triangle (12.5) 


0.03 


0.75-0.90 




Disk (9.0) 


15 


13 


Inverted triangle 


0.14 


0.50-0.75 










(12.5) 





















i. indicates maximum test diameter or diameter of disk model, cm. 
ii dii ates length of single side of model, cm. 



HOST RECOGNITION BY SHRIMP 105 

most shrimps within the 6.0 minute test period. Table V summarizes the results 
of these investigations. 

To determine whether urchins are discriminated hy size, shrimps were per- 
mitted to choose between a moderate and a larger-sized urchin, as well as between 
the same moderate-sized urchin and a smaller one (Experiment 1). In each case, 
the cell containing the larger urchin of the pair was favored. To test the possibility 
that larger shrimps preferentially choose larger urchins, a two-way contingency 
analysis was applied to the number of small (< 1.5 cm total length) and large 
(> 2.5 cm total length) shrimps choosing the larger of the two hosts in each 
pairing. Moderate sized shrimps were not included in this calculation in order to 
produce more discrete size classes of small and large individuals. For both pair- 
ings, the hypothesis of no difference in response between small and large sized 
shrimps could not be rejected ( x 2 - 1.82. 1.56; P - 0.10-0.25). 

To determine if urchins are recognized by the presence of spines, shrimps were 
permitted to choose between a large urchin (8.0 cm test diameter) from which 
all spines had been clipped off to within 0.5 cm of the test and a small urchin (4.0 
cm test diameter) from which only the tips of the longest spines had been clipped 
to obtain a peripheral diameter of 9.0 cm (Experiment 2). Both "urchins" had 
Ihe same peripheral diameter thus minimizing experimental bias due to a size dif- 
ference between the two test objects. If the presence of spines or the spinose 
form was an attractive parameter, shrimps should favor the smaller of the two 
urchins, i.e., the one with spines essentially intact. Neither cell was favored, 
however. If spines are not necessary for urchin recognition, it should further be 
possible to construct a solid dark colored model that could not be differentiated 
from an intact urchin of equal effective visual diameter. Two-dimensional models 
fashioned from thin sheet plastic and painted flat black were presented by mount- 
ing them on a panel of clear plastic centered vertically in the test cell. Since it 
was not possible a priori to equate disk diameter with urchin peripheral diameter, 
a series of three pairings was conducted in which shrimps were permitted to choose 
between a 10 cm diameter black disk and one of three different sized urchins 
(Experiment 3). No discrimination was obtained in favor of either cell when 
intact urchins of 7.5 and 5.0 cm test diameter were paired with the disk. \Yith 
a more extreme size differential, a 10 cm disk vs. a 3.5 cm test diameter urchin, 
however, the cell containing the solid disk was favored. 

A third experiment was directed towards evaluating spines as a parameter of 
recognition. Shrimps were permitted to choose between a 9.0 cm diameter solid 
black disk and a 9.0 cm diameter patterned black-white disk consisting of a 
"checker-board" of alternating black and white squares 1.0 cm on a side (Ex- 
periment 4). If shrimps recognize the spinose form of the urchin on the basis 
of the internal contrast, the patterned black-white disk, with its greater internal 
contrast, should be favored over the solid disk of equal peripheral diameter. The 
cell containing the solid disk was favored over that containing the patterned disk. 

To determine if urchins are differentiated as a discrete form per se or as a 
contrasting pattern with the background, the transparent "windows" of the test 
cells were masked with opaque screens. The size of a disk was calculated so that 
its total area equaled one-half the area of the opaque screens and a disk centered 
on each screen. Shrimps were permitted to distinguish between a white disk 



106 BARRY W. ACHK AND DKMOREST DAVENPORT 

presented against a black background and black disk presented against a white 
background (Experiment 5). Both models contain equal areas of black and 
white. Both contain equal /one.- of black -white boundary. Both models should be 
equally attractive if shrimps are responding to the amount of light-dark contrast. 
The cell containing the black disk against the white background was favored over 
the reverse combination. 

To determine if urchins are differentiated on the basis of their peripheral out- 
line, shrimps were permitted to choose between a 9.0 cm diameter disk and a 
series of two dimensional shapes (Experiment 6). A 9.0 cm diameter solid black 
disk was favored over a 9.0 cm diameter serrated black disk with 28 equally- 
spaced serrations cut radially to a depth of 2.0 cm. Neither cell was favored on 
presentation of a solid, black square. 7.9 cm on a side, and a c ).0 cm diameter solid 
disk, these models enclosing equal areas. However, if the size of the square 
shape was increased so that its area equaled 1.3 times that of the 9.0 cm diameter 
disk (i.e.. 8. ( } cm per side"), the shrimps favored the cell containing the square 
shape. To control the possibility that the overall size of the models exceeded 
the visual angle subtended by the shrimps' eyes, the square-disk pairing was again 
presented except that the area was reduced and equated to that of a 6.0 cm 
diameter disk (square 5.3 cm per side). As with the larger models, neither cell 
was favored in this latter pairing. Finally, shrimps did not discriminate between 
9.0 cm diameter solid black disk and a solid black equilateral triangle of equal 
area (12.5 cm per side), either with the triangle oriented point upward or inverted 
with the point downward. Experiment (> supports the conclusion that visual recog- 
nition of the host urchin, .V. jranciscanus, by the shrimps is not based on the per- 
ception of the urchin as a round or circular form. 

DISCUSSION 

The data support the hypothesis that B. Jiarfordi is able to effect distant host 
recognition utilizing chemical stimuli of host origin alone. It is not suggested 
that visual cues are entirely without effect when available. Indeed, Experi- 
ments 1-3 and 24- (Table II) suggest that visual stimuli of host origin may 
elicit a low response under certain conditions. However, both these apparent 
responses to visual stimuli can also be explained as a generalized response 
toward the only contrasting object in an otherwise monotonous choice situation. 
The results of Experiment 2-2 (Table II) which incorporate essentially the same 
stimulus parameters as Experiment 1-3 (Table H) did not indicate any tendency 
of the shrimp to select the model-containing cell. It is interesting to note that 
subtidal species of Haliolis are frequently heavily encrusted with epiphytic 
growths and contrast little with the surrounding substrate (Cox. 1962). How- 
ever, the black epipodium of at least one of the subtidal species, H. nijcsccns. 
could olter sufficient contrast with the background to facilitate visual recognition 
at close range. 

A question arises as to the ability of chemical stimuli alone to effect directed 
locomotion towards an odor source (Fraenkel and Gunn. 1961; Gage, 1966). 
In the marine benthos local water turbulence and surging would disrupt diffusion 

dients required for chemotaxic orientation. It appears that B. Jiarfordi can 
the directional component of a carrier current containing host factor to 



HOST RECOGNITION BY SHRIMP 107 

effect host location by moving "upstream" in the presence of the appropriate 
chemical releaser. This mechanism reportedly occurs in other crustaceans (Alice, 
1916; Luther, 1930), although to the authors' knowledge it has not been investi- 
gated in any detail. Laverack ( 1962) has demonstrated low frequency tactile 
receptors in Hoinarus chelae, sensitive to water currents down to 0.3 cm sec. 
Low threshold chemoreceptors have been well documented in the Crustacea (e.y., 
review of Laverack, 1968). Such a mechanism would be adaptive in that it would 
not require chemoreceptor competence sufficient to discriminate the very small 
increments in chemical concentration required for chemotaxic orientation, but 
merely the presence or absence of the attractant. 

Specificity experiments suggest that B. harfordi discriminates a chemical sub- 
stance or complex of substances containing sufficient information for recognizing 
gastropods of the genus H allot Is from other gastropods and from the non-molluscan 
hosts of other betaeid shrimps. The data do not eliminate the possibility that this 
chemically-mediated genus-specific recognition is based on quantitative rather 
than qualitative differences in the attractants. The fact that equal masses of test 
organisms were contained directly in a continuously flowing seawater wash mini- 
mizes experimentally induced variations in stimulus concentration, so even quanti- 
tative differences in the same attractant must be considered as potentially significant 
mechanism for maintenance of this association. Recently, evidence has been pre- 
sented that quantitative odor differences are at least partially responsible for 
mediating escape/attack behavior in a marine gastropod (Snyder and Snyder, 
1971). 

Previous investigations indicate a relative high degree of chemosensory com- 
petence in crustaceans. Symbiotic pinnotherids, P'mnixa chaetopterana, dis- 
criminate effluents of host polychaetes of the genera Chactopterus and AuipJiritritc 
from those of the non-host polychaete genera, Nereis and Arenlcola (Davenport 
et al., 1960). Another symbiotic pinnotherid, Dissodactylus mellitac, discriminates 
its host echinoid. Mellita quinquiesperforata^ from six other species of echinoderms, 
although it reportedly can be conditioned to respond to another flattened echinoid, 
Encopc uiitchelini (Gray et al., 1968). More specific discrimination has been re- 
ported (Carton, 1968) for the parasitic copepod Sabelliphilus sarsi which can dis- 
criminate by chemical means between its host polychaete, Spirographis spallanzani 
and two non-host but congeneric polychaetes, S. pavonina and S. spallanza-ni var. 
brei'ispim. By nature of their action, crustacean sex phermones (Atema and Eng- 
strom, 1971 ; Kittredge, Terry and Takahashi, 1971 ; Ryan, 1966) could also be 
considered species-specific chemical attractants, but the possibility that other 
stimulus modalities confer the species specificity to crustacean chemically-mediated 
mate recognition remains to be disproven. 

B. inacginitieae, in contrast to B. harfordi, appears to use both visually and 
chemically mediated information for distant host recognition. The interaction of 
chemical and current stimuli was not adequately investigated for B. niacginitieae, 
and thus is not reported here. Visually mediated information would contain suf- 
ficient directionality, however, to allow directed locomotion in situations offering 
both chemical and visual stimuli, even in the absence of current flow. It is possible 
that chemical and visual cues, acting together enhance the value of the stimulus 
situation to the shrimps. More shrimps located the host urchins in the arena 



108 BARRY W. ACHE AND DEMOREST DAVENPORT 

experiment presenting both stimuli simultaneously than in those lacking either 
visual or chemical cues (Table III, Experiments 1, 2, 3). Such apparent enchance- 
ment does not necessarily result from neural summation of the sensory information 
contained in the two stimulus modalities, however, since it could also be explained 
by chemically released hyperactivity increasing the probability that visually directed 
locomotion towards the host occurs within the test period. The data do not allow 
resolution of this question, but the phenomenon is worthy of further investigation. 
Symons (1964) earlier reported that the number of feeding movements nearly 
doubled in the crab Hemigrapsus oregonensis when elicited by both chemical and 
tactile stimuli together than by either stimulus modality operating alone. 

The specificity of the Betaeus-Strongylocentrotus association in nature is 
somewhat unclear. Hart (1964) described the species with nine specimens, one 
pair collected from .V. purpnnitus, one female from 5\ franciscanus, and six with no 
host record. In an area abundant with both urchin species, over 1000 specimens 
of B. mact/initieae were collected, associated in all but one instance with speci- 
mens of i\ franciscanus. This fact, along with the fact that B. macginitieae is 
homochromous with .V. fraud sea nits and behaviorally adapted to move among the 
long spines of this urchin (Ache, 1970) suggests that S. franciscanus may be the 
"preferred" host of B. macginitieae and S. pitrf>iiratits a secondary host. 

Chemical stimuli contain sufficient information to allow the urchin symbionts to 
discriminate urchins of the genus Strongylocentrotus from other echinoderms 
and from the non-echinoderm hosts of other betaeid shrimps, a level of sensory 
competence at least functionally similar to that of B. liarfordi. Visually mediated 
information is sufficient to allow B. macginitieae to further discriminate between 
the two reported strongylocentrotid hosts. The long-spined, brick red (occasionally 
to light red) .S". franciscanus is morphologically distinct from the short-spined, 
smaller, light purple S. purf^nratns (Ricketts and Calvin, 1968). Thus B. macgini- 
tieae with its demonstrated ability to visually discriminate large, dark "solid" objects 
could differentiate between the two urchin species. Visually-mediated behavior is not 
commonly reported to occur in aquatic crustaceans, although its role is rather well 
documented in the control of sexual and agonistic behavior of semi-terrestrial species 
(e.g., reviews of Schone. 1968; Salmon and Atsaides. 1968; Wright, 1968). Alver- 
des (1930) noted that the aquatic branchyuran Carcinus inacnas and the anomuran 
Iiitpai/itnis bernhardns confronted by two black screens will move between them, 
but before doing so beat their antennae in the direction of each of the screens, 
behavior he interpreted as suggesting that perception of the screen as objects does 
occur. Visually-mediated food location behavior has been reported for the aquatic 
anomuran Clibanarius fit fat us ( Hazelett, 1968) and the intertidal brachyuran 
/'acliy</nif>sus cnissipes ( Hiatt, l ( H8i although these latter observations were con- 
ducted on crabs in air. Symons (1964) was unable to demonstrate either a 
releasing or directing effect of visually-mediated stimuli on the feeding behavior 
of the aquatic brachyuran H* -psus orci/tnicnsis. 

Ouestion arises as to the ability of visual stimuli acting alone to effect a 
response specific to \. franc if B. macginitieae is not responding to any 

visual parameter uniquely cha of its host. Experiments showed that the 

predominately black hemispherical Mrr/athura was not distinguished from the 
host .V. franciscanus in a paired ch< .situation, although visual stimuli proved 



HOST RECOGNITION BY SHRIMP 109 

sufficient for discrimination of the host urchin from other lighter-pigmented 
organisms. The possibility must be considered that few other large, dark-pig- 
mented organisms like J\lc(/athnra may occur in the subtidal rocky habitat of the 
range ascribed by Hart for the Betaeus-Strongylocentrotus association (Santa 
Catalina Island, Monterey, California). Further experimentation, however, is 
necessary to clarify this point. 

A visual receptor capable of rudimentary form vision would be sufficient to 
effect the visually-mediated behavior demonstrated by the shrimps. As noted by 
Carthy (1958), what appears to be simple form recognition of dark shapes can 
frequently be explained by the alternative hypothesis of a negative phototaxis 
towards a zone of reduced light intensity. That a more complex response than 
simple negative phototaxis is involved is demonstrated by the preference of 
B. macginitieae for the black circle presented against a white background over the 
white circle presented against a black background (Table V, Experiment 5 ). Both 
models presented equal zones of contrast and equal areas of reduced intensity. 
This is not to imply that the shrimps are not negatively phototactic ; it has been 
shown they are (Ache, 1970). The peripheral outline of the model does not 
appear to be an active parameter in discrimination (Table V, Experiment 6) 
suggesting that the attractive factor may be more the "solidness" of the form than 
its specific shape e.g., circular or semi-elliptical as the urchin test. The preference 
of shrimps for the solid circle over the black-white checkerboard-patterned model 
(Table V, Experiment 4 ) also supports this idea. 

The possibility of color discrimination has not been eliminated by these experi- 
ments. Two factors tend to discredit the possibility that .S\ franciscanus is recog- 
nized on the basis of color. Black models proved as equally attractive as naturally 
pigmented urchins, when equated for effective visual diameter (Table V, Experi- 
ment 3). Also the extinction coefficients of coastal seawaters are greater for longer 
wavelengths of visible light required for color discrimination of a red pigmented 
organism (for coastal water off Southern California- Young and Gordon, 1939). 
However, Wald and Seldin (1968) have demonstrated differential sensitivity of 
two components of the ERG in the shrimp Palaemontes i'iil(/aris which they sug- 
gest may represent the red- and violet-sensitive components of a visual mechanism 
for color differentiation. The results of the present experiments, however, indicate 
that intensity discrimination would be sufficient to explain the visually-mediated 
component of host recognition. 

It appears then that information from several sensory modalities is utilized by 
both B. harfordi and B. macginitieae to effect their respective symbiotic relation- 
ships. This mechanism serves to reduce the demands on the competence of any 
one receptor type, while maximizing the discriminating ability of the shrimps both 
in regards to stimulus directionality (the B. harfordi studies) and stimulus 
specificity (the B. macginitieae studies). The present experiments do not allow 
resolution of whether the action of such multi-modal information is simply addi- 
tive or involves summation and perhaps additional integration in higher neural 
centers. Certainly, centers of higher order neural integration receiving visual in- 
formation and input from other sensory modalities, including chemosensory in- 
formation, are known to exist in the eyestalks of Panulirus argns (Maynard and 
Dingle, 1963; Maynard and Yager, 1968) and probably in other decapod species 



110 BARRY W. ACHE AND DEFOREST DAVENPORT 

(Hazlett, 1971). Work towards resloving this question is currently in progress 
using crustaceans of several species. 

SUMMARY 

1. The sensory basis of host-oriented locomotion in the caridean Bctacns 
macginitieae contrasts with that of the congeneric />. Jiarfonfi. Both of these 
shrimps can locate their respective host organisms utilizing chemical stimuli of 
host origin. Only B. inttci/iiiiticac demonstrates the ability to utilize visual stimuli 
for this same purpose. 

2. By using information contained in multiple stimulus modalities, B. macgini- 
tieae is able to maintain a more restricted host association than its congener. 

3. Visual recognition of its urchin host by B. macginitieae does not involve any 
parameter of the total visual pattern of the urchin that uniquely characterizes the 
urchin species, but appears to be a generalized response to larger, dark forms of 
undefined peripheral outline. 

4. Positive rheotaxis in the presence of appropriate non-directional chemical 
stimuli is suggested as the mechanism by which B. harfordi effects chemically- 
mediated host location. 

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VISUAL ORIENTATION AT THE WATER SURFACE BY 
THE TELEOST ZEX ARCH OPT ERUS x 

RICHARD B. FORWARD, JR., 2 KENNETH W. HORCH 3 
A .XT) TALBOT H. WATERMAN 

Biology Department, Yule Vnircrsity. AYri- Ihnrn. Connecticut 06520 

In seeking quantitative documentation that certain teleosts can perceive the 
e -vector of linearly polarized light (earlier work reviewed in Waterman, 1972), a 
number of field experiments have been carried out on the viviparous tropical half- 
beak Zenarchopterus (Hemirhamphidae). Most of these were underwater studies 
conducted with single fish enclosed in a covered transparent vessel and exposed to 
various illumination patterns including both natural and imposed polarized light 
(Waterman and Forward. 1070; 1972). 

Although halfbeaks were behaviorally responsive in this submerged situation, 
they normally swim at the water surface. In order to include this potentially im- 
portant feature of the fish's visual and tactile environment, additional experiments 
were conducted on land with the fish swimming in an open experimental vessel. 
Under such conditions a fairly vigorous basic polarotaxis was evoked that w r as 
somewhat different from that previously observed. Also new evidence was found 
relating to other components in the fish's visual orientation. These studies of 
Zenarchopterus? visually evoked directional behavior at the water-air interface are 
the subject of the present report. 

To begin with some precise definitions of terms describing animal orientation 
to light may be helpful. Phototaxis is the directionally oriented response of an 
organism to light intensity patterns (Kuhn. 1919; Fraenkel and Gunn, 1940; 
Jander, 1970). The comparable response to light polarization patterns is polaro- 
taxis (Waterman, 1966), i.e., a directional response in relation to a given plane 
or pattern of polarization. When the resulting orientation (body axis alignment 
or steering direction of locomotion) has a fixed, non-graded angular relation to 
the stimulus, e.g., heading toward the light (0) or away from it (180) (photo- 
taxis), or at 0, 45, 90, 135 to the ^-vector (polarotaxis), then it may also be 
called a "basic" response or basita.ris (== "basotaxis" of Jander, 1963a, 1963b). 

In contrast to basitaxis there is another relatively simple type of response to 
spatial differences in light intensity. This is photomenotaxis, or the light compass 
reaction (von Buddenbrock, 1917), in which orientation may be at any temporarily 
fixed angle to the stimulus source. A comparable compass orientation depending on 
linearly polarized light is also well known in many arthropods (Waterman, 1966). 

For behaviorally significant direction-finding which utilizes celestial cues, a 
menotaxis rather than a basitaxis would ordinarily be needed, since the course 

1 This research was supported by grants from the U. S. Public Health Service, tin- 
National Geographic Society and the National Aeronautics and Space Administration. 

2 Present Address: Duke University Marine Laboratory, Beaufort, North Carolina 28516. 

3 Present Address: Department of Physiology. University of Utah, Salt Lake City, 
; 84112. 

112 



VISUAL ORIENTATION BY ZENARCHOPTERUS 113 

heading required would depend on the animal's present position relative to the 
location of its "goal." Thus as the animal moves the angular relationship between 
the compass direction connecting these two points and the reference cue will usually 
vary with time. Moreover, if the orientation is to he accurately maintained by 
celestial reference for more than a few minutes, the compass reaction would have 
to be time compensated to allow for the apparent movement of the sky and celestial 
bodies as the earth rotates (von Frisch, 1950 ; Kramer, 1950). 

Fishes have been shown to be capable of a time-compensated sun compass 
orientation (reviews: Hasler, 1966; Harden Jones, 1968; Waterman. 1972). 
This ability implies that from their underwater vantage point fishes are able to 
determine the sun's azimuth direction and possibly its altitude. Close to the surface 
and in a flat calm this might be accomplished by direct observation of the sun's 
disc or sky polarization. Otherwise it could be derived either from the radiance 
distribution in the water or from the underwater polarization pattern, both of 
which are directly dependent on the sun's position (Waterman, 1954; Jerlov, 
1968;Lundgren. fo/1). 

The light intensity distribution is probably suitable for accurate localization 
(within say 6) in shallow water (5-10 in), However, the useful information 
Irom this radiance pattern decreases rapidlv with increasing depth (Harden Jones, 
1968). A more precise indicator of the sun's position which penetrates to greater 
depths is the polarization pattern underwater (Waterman, 1955; Tvanoff and 
Waterman, 1958). Therefore, demonstration of polarotaxis by fish would imply 
that they are capable of perceiving this component of natural submarine illumina- 
tion. In turn this suggests that they could use the underwater polarization pattern 
to localize the sun. 

We present herewith new evidence for polarotaxis as well as for time com- 
pensated sun compass orientation by the fish Zenarochopterus. 

EXPERIMENTAL METHODS 

The experiments were carried out in Palau, Western Caroline Islands (U. S. 
Trust Territory of the Pacific Islands) on August 28th and 29th, 1970. The 
general methods employed resemble those for the corresponding underwater studies 
( Waterman and Forward, 1972) except that the fish were tested in a transparent 
vessel open to the air with a free water surface. 

The experimental site was the broad cement apron of an abandoned seaplane 
ramp on the north shore of Arakabesan Island (E, Fig. 1). Single fish were 
placed in a cylindrical transparent plastic container, 19 cm in diameter and 7.5 cm 
deep. The vessel was positioned above an intervalometer-controlled Robot camera, 
and screened laterally and downward by white, cylindrical screens and a diaphragm. 
Thus, the fish had a free view of the sky through the water surface, but was 
prevented from seeing surrounding landmarks, the experimenters or the equip- 
ment (except for the camera lens). To minimize reflection from the meniscus 
or from the cylinder's plastic wall in air, the vessel was carefully filled with 
seawater just to its brim. Freshly caught, experimentally naive juvenile Zcn- 
archopterus dispar (Cuvier and Valenciennes) 40-50 mm in length were used. 
All fish were collected at a localized site on Auluptagel Island (H, Fig. 1). 

During the experimental period, the sun's bearing and elevation were deter- 



114 



R. B. FORWARD, JR., K. W. HORCH, AND T. H. WATERMAN 



I3427 E 



28 



29' 



' 




21 




7 20 

N 






ARAKABESAN 
ISLAND 



i 



331 



Aug. 29 



;* 

;.X 

X 



: - 
2^-361 .'..' '.ox* 

** mm \ 1 1 ' 

: .\ ? ' I* 

^ 

- ' 



iX 







-21' 



: KOROR ISLAND. :.'. 



i 05 V-V:V: :':-. 




134 27 E 



28' 



720 

N 



29 



Fici'KE 1. Map of Arakabesan, Auluptagel, and nearby area of the Palau Islands. The 
experimental site was at E and all the Zenarchopterus used were collected at H. The double 
headed arrow near the latter indicates the main trend of the inter-island channel adjacent to 
// (channel axis 150-330). Island peak elevations are given in feet. Outer edge of coral 
reefs are represented by scalloped outlines, mangrove areas by cross-hatching. 



mined every 10 iiiin with compass and sextant (Fig. 2). To document cloud cover 
conditions, photographs of the sky were taken at similar intervals with a camera 
having a fisheye lens. The sky generally was 35-40% obscured by light scattered 
clouds on the two days concerned. However, fish orientation was recorded only 
when the sun was clearly visible. When a small cloud briefly obscured the sun, 
measurements were suspended until the sun was again shining. 

Twenty-four specimens of Zenarchopterus were studied, each under four optical 
conditions: (1) without the polarizer (NF condition), during which the fish was 
exposed only to the natural illumination of sun and sky ; and (2, 3 and 4) with a 
Polaroid linear polarizer (Type KN36) placed over the experimental vessel (WP 

'lit ion ) .11 id oriented at three different directions with respect to the sun's bearing 
3). Since six planes of polarization were tested altogether (0-180, 30-210, 



VISUAL ORIENTATION BY ZENARCHOPTERUS 



115 



60-240, 90-270, 120-300, 150-330), only half were used with an individual 
fish. 

For each condition, 10 consecutive photographs of the fish's headings were made 
at 10 sec intervals timed and counted by the intervalometer. The filter was then 
changed manually to its next position or removed for the NF condition. After 
a 10 sec pause to let the fish settle down, another 10 frame sequence was made, 
etc. The order of presentation of the conditions, as well as the angle of the 
polarizer were randomly selected with the constraints that no condition was used 
twice with a given fish, and all conditions were tested an equal number of times. 

The angle between the fish's longitudinal axis and the sun's bearing taken as 
reference was measured to the nearest 10 from the photographs. For analysis 
these headings were grouped into 30 sectors centered to coincide with the six 



120 



CD 60 

Z 

tr 
LU 

CD 

CO 



300' 



240 



i i r 



l r 



i i 




w 



_L 



l 



90 C 



60' 



CO 



CO 



so : 



0" o 
m 



to 



AM 



12 



PM 
LOCAL 



14 
T 



16 



18 



M E 



FIGURE 2. Sun's hearing and altitude during the experimental periods. Note that for 
August 28th the solar bearing \vas constant near W by WNW during the full period. On 
the 29th the bearing was stable for the first 2.5 hours, then it shifted very rapidly. For this 
reason measurements made during the last 30 min (4 of the 12 fish) were excluded from the 
main analysis. 

polarization planes tested. Obviously the positions and angular extent of these 
sectors are fixed and the response variable is the number or percentage of observed 
orientations which falls within each. 

The experimental design permits the overall data to be analyzed in several ways 
to test for various significant stimulus components. Thus the total distribution as 
recorded has the sun's bearing at and the imposed ('-vector randomized (Fig. 3). 
When transposed to geographical coordinates (0 -North) the r -vector is still 
at random but the sun's bearing appears mainly in two opposite directions. Their 
effect can be determined by separating the data for days. 

The NF data can be compared with the randomized WP distribution to check 
for possible behavioral changes due to other features of the polarizer than its e- 



116 R. P>. FORWARD, JR., K. W. HORCH, AND T. H. WATERMAN 

vector orientation. Then the YY P counts can be transposed so that all the im- 
posed c-vectors are aligned with the 0-1SO plotting axis. This distributes the 
influence of the sun and rny geographically derived effects leaving just polarotaxis 
as the predominant source of orientation in the resulting pattern. Finally the 
NF data can be compared with those for any particular imposed r-vector orienta- 
tion. 

To begin with the responses of the 24 individual fishes were studied in detail. 
Previous experience showed that "inattentive" fish significantly decrease the overall 
response level to potential orienting stimuli (\Yaternian and Forward, 1972). 
Obviously a completely inactive fish and one orienting consistently "on the beam" 
will both maintain constant headings. However, the inattentive animal can be 
recognized by its failure to change headings over a long period even when the 



P-60 



P-30 



Sun 
Aug 28 



P-0 



P-150 




P-150 



P-0' 



P-30' 



Sun 
Aug 29 



P-120 



P-60 



P-90 
South 

FIGURE 3. Angular relationship between North, sun's azimuth 
and polarixer r-vector directions (P-0, etc.). 

experimental conditions are altered. Inattention may also appear as a continuously 
changing orientation at a constant or high velocity. In such cases no externally 
cued directional preferences may be involved. 

The major results presented below are derived from data selected to minimize 
these difficulties. However, the effects of such selection have been repeatedly 
checked by comparisons with the total data and with distributions resulting from 
alternative selective procedures. For example the last four fish run on August 29 
were eliminated from prime consideration because the sun was changing its bear- 
ing very rapidly during their runs (Fig. 2). Also at that time the solar zenith 
distance, and hence the sun's potential influence in determining azimuth, was mini- 
mal as was the sky polarization near the zenith. 

Indeed these fish showed different but les coherent orientation preferences 
than those run with steady sun's bearings. Nevertheless addition of their counts 



VISUAL ORIENTATION BY ZENARCHOPTERUS H7 

to those of the other 18 orienting fish does not alter the location of significant 
sectors relative to the North, sun, or r-vector. Nor do they significantly affect the 
overall NF distribution. 

Furthermore two other fish from the afternoon sequence were eliminated from 
the selected data because they scarcely changed their headings throughout the set 
of four conditions tested for each. Hence 18 of the total 24 fish provide the orienta- 
tion headings analyzed in most detail. 

Moreover for all fish rather large angular changes of direction sometimes did 
occur between frames. To decrease the influence of such more rapid turning, we 
have for our selected data rejected orientation measurements which differed from 
their predecessor by more than 20. The remaining headings are interpreted as 
"pauses" in directions preferred by the fish and possibly determined by some ex- 
ternal clue for azimuth. 

Since behavior patterns of the individual fish were different the number of 
pauses observed under each experimental condition varied. Therefore, to pre- 
vent over or under representation by any one condition when totaling the results. 
the data for each condition were converted into percentage responses in each 30 
sector. Where different conditions are combined, the percentage responses in each 
angular category are totaled rind divided by the number of conditions used. 

Ninety-nine per cent binomial confidence limits were computed for the ob- 
served frequency of responses in each sector. If the expected frequency for a uni- 
form distribution (c.y., 8.3% in each 30 sector of a circle) fell within these limits, 
the observed frequency is considered not significantly different (at the \% level) 
from a random, non-oriented response. Otherwise, it is indicated in the figures 
as being above (plus sign) or below (minus sign) the expected value. 

Note that this treatment was used instead of the attractive circular normal 
method ( Batschelet, 1965; Waterman and Forward, 1970) because our data are 
generally not the normal, unimodal, continuous distributions appropriate to the 
latter. Furthermore most of our comparisons are made with normalized relative 
frequencies, not with the actual counts. 

RESULTS 

For all 24 fish on the two days 933 heading counts are available including all 
seven conditions ; the corresponding number for the pauses, as defined above, is 
636. For the 18 selected fish which were orienting while the sun's bearing was 
steady the total data are 775, the pauses 444. Consider first the circular distribu- 
tion of the 18 fish on two days summed for all conditions (WP and NF) and 
plotted relative to North (Fig. 4A). Here all three sectors in the southerly quad- 
rant are significantly preferred as is the sector centered at 30. 

Examination of the detailed data shows that the last sector's significance depends 
mainly on several runs on the afternoon of August 28. The pause criterion elimi- 
nates these counts, however, while reinforcing the southward preference (Fig. 4B ). 
Indeed the pause count in the 30 sector in Figure 4P> is significantly less than 
expected as are those in the other sectors from North to West. We can conclude 
then that the peak near NNE is behaviorally distinct as it is associated with more 
rapid turning by the halfbeaks. The broad southerly preference is characteristic 
of both the total counts and the pauses. 



118 



R. B. FORWARD, JR., K. W. HORCH, AND T. H. WATERMAN 



330 




240 



20% \90 



120 



210 



150 



ISO 



180" 



FIGKRE 4. Zc>i(ircli<>f>tt*nis' directional responses relative to North and to the sun. A and 
B permit comparison of total (n 775) and pause (n = 444) data (see text for definitions) 
plotted relative to North at 0. B and C (11 = 444) compare the pause data distributions 
relative respectively to North and to the sun's bearing. Eighteen fish, both days, seven con- 
ditions (one without polarizer and six different r-vector directions). Plotted as percentages 
of the numbers observed in twelve 30 sectors around 360. Directions whose sectors con- 
tain greater percentages than expected at the 99% confidence level are indicated by "+" and 
those having less than expected by " ." The distribution in Figure 4C is dissected by days 
in Figure 5. 

To analyze this geographic directional choice further the data need to be con- 
sidered relative to the sun's bearing. When so plotted the pause distribution 
obtained for the IS selected fish shows only one significant preferred sector at 150 
clockwise from the sun's bearing (Fig. 4C). However, note that the whole 
(|uadrant centered in the solar direction is significantly avoided. 

Since the sun's hearing differed by nearly 180 on the two experimental days 

' l ; ig. 2) this overall distribution needs to be dissected accordingly. The cor- 

onding orientation patterns are quite different (Fig. 5A, B). Both are 

il a:id skewed away from the sun. But in the morning data (Fig. 5B) 



VISUAL ORIENTATION BY ZENARCHOPTERUS 



119 



the significant sectors are at 60, 120 and 150 re the sun's bearing. Geograph- 
ically these range from about SSE through SW. In contrast the afternoon data 
(Fig. 5A) show peaks at 240, 270 and 300. But their geographical distribu- 
tions are about SSW, S and SSE. On both days the quadrant centered on the 
sun's bearing was significantly avoided as was one other quadrant which refer- 
ence to the geographic coordinates identifies as northerly for each case. The 
apparent avoidance of the sun's general direction and the broad southward tendency 
which is shown by a preponderance of headings 90 to the sun's vertical indicate 
that the solar azimuth is involved in the observed responses. 

These implications are strengthened by the oriented reactions to the NF con- 
dition where the fish were responding without a filter-imposed r-vector (Fig. 6A). 
Some counts appear in all but one sector (300 re N) but the only direction 



30' 



300 



270- 



240 




210 



180 



FIGURE 5. Comparison of Zenarchopterus' orientation relative to the sun on the two 
experimental days; (A.) August 28, 10 fish, n = 253 ; (B.) August 29, 8 fish, n = 191 ; 
pauses only, seven conditions (one without polarizer, six with different e-vector directions). 
Sectors and significant directions as in Figure 4. Figure 4C is the sum of the data in 
Figures 5A and SB. 

significantly preferred was 180 (South). In addition the southerly quadrant was 
preferred in 4S c /c of the pause headings. Examination of the corresponding total 
count distribution for the 18 fish shows a similar pattern except that an additional 
significant peak is present at 30. 

We have already seen that this is associated with faster turning and is eliminated 
by the pause criterion. The 24 fish total count distribution like the 18 fish pause 
count for NF has only one significant sector and that, too, is at 180 ; the last 
four fish on the 29th reacting while the sun's bearing was changing rapidly showed 
no significant peaks in their NF distribution. Thus a strong southward orienta- 
tion was evident in our Zenarchopterus data not only with the six WP conditions 
(randomized - vector) plus NF but in the last condition alone where just the 
natural illumination was available. 

Of course the blue, skv visible in the NF condition has its own characteristic 



120 



R. B. FORWARD, JR., K. \Y. HORCH. AND T. H. WATERMAN 



polarization pattern. A compan'smi between the NF response and that WP 
condition most closely simulating the natural pattern should therefore be instruc- 
tive. The sky polarization in the zenith and all along the vertical great circle 
passing through the sun, i.e., the sun's vertical, is perpendicular to the solar bear- 
ing. Consequently the 90 WP condition of the six used would no doubt most 
closely resemble the natural skv pattern. 

In the latter, however, the degree of polarization, maximum about 90 from the 
sun, varies for blue sky from 50-90% depending on the earth's albedo, the turbidity 
of the atmosphere and the sun's zenith distance (Sekera, 1957). Near the surface, 
if the water is relatively calm as in our experimental vessel, this polarization as 



270 



240 



120 




240 



120 



210 



150 



180 



Comparison of Zcnarchoptcrus' orientation relative to North under natural 

Humiliation and with different c-vector directions imposed by a polarizer; (A.) natural 

Humiliation without polarizer (NF condition), n = 96 ; (B.) with polarization plane at 90" 

sun's bearing (parallel to natural polarization in the sun's vertical), n = 53 ; (C.) with- 

r runs for five of the six specific c-vcctor directions (WP at 0, 30, 60, 120, 150). 

(Fig. 6B) is excluded here. See text for discussion, n = 295 ; Eighteen 

Sectors and significant directons as in Figure 4. 



VISUAL ORIENTATION BY ZENARCHOPTERUS 

well as that of the whole sky would be directly observed underwater through the 
critical angle (Waterman, 1954). In contrast our experimental 90 WP condi- 
tion provided nearly 100% polarization of the visible light with the r-vector 
perpendicular to the sun's bearing apparent through the whole sky not just along 
the sun's vertical. 

The corresponding halfbeak orientation with 90 \YP indicates that more than 
half of the sectors were avoided and the three southward ones (approximately 
SSE, S and SSW) were significantly preferred at better than the \% level 
(Fig. 6B). Indeed S2'/c of the headings in this distribution fall within the 
southerly quadrant. 

Note that the approximately 65% reduction in luminous flux due to placing 
the polarizer over the experimental vessel did not alter the fishes' general be- 
havior or azimuth preference when the imposed r-vector was parallel to that of 
the sky in the sun's vertical. Actually the resulting increases in the degree of 
polarization and the extension over the whole celestial hemisphere of the X-S 
r-vector direction naturally present in the sun's vertical are correlated with a 
reinforcement of the southward preference. 

Because rather similar strong southerly preferences were demonstrated by the 
XK and 90 WP data one could argue that these two conditions (out of the seven 
tested) were sufficient to dominate the overall data distribution relative to geo- 
graphical North (Fig. 4A, B). This is not the case, however. If all the pause 
data minus the NF and 90 runs are plotted, 44% of the headings are still in the 
quadrant centered on S (Fig. 6C). This is a significant preference over random 
for the four quadrants at better than the 1% level. The same was true of course 
for the NF and 90 WP plots themselves (Fig. 6A, B) but this tendency persists 
in the rest of the data, too (Fig. 6C). Therefore we can conclude unambiguously 
that the southward preference indicated in the total \VP plus NF pauses is not 
due to the NF and 90 runs alone. 

To test for any polarotactic effect of the imposed ^-vector all responses to the 
six geographical directions of polarization plane produced by the Polaroid filter 
should be appropriately grouped and totaled. To permit this data were "zero 
corrected" by transposing counts so that the r-vector directions for all six WP 
conditions fall on the 0-180 plotting axis. Since polarized light is symmetrical 
about the r-vector axis, diametric responses, e.g., 90 and 270 are equivalent 
(Fig. 2). Thus, if it perceives the polarization plane an organism can tell 
whether its anteroposterior axis is heading to the right or left of this r-vector. 
However, on the basis of that clue alone orientation would be subject to an 180 
ambiguity around the horizon. 

Heading responses were therefore folded around the axis perpendicular to 
the plane of polarization by adding diagonal pause counts. This yields a semi- 
circular distribution counterclockwise (left) and clockwise (right) from the c- 
vector. Corrected in this way, a fairly strong and significant (at the 1% level) 
preference is shown for the direction, i.e., parallel to the ^-vector (Fig. 7A). 

The validity of this conclusion can be reinforced by showing that the selec- 
tion of data for pauses sharpens the decided preference of Zenarchopterus for 
orientation parallel to the ^-vector. Yet it does not alter the inferences which 
may be just as clearly drawn from the unselected total data for the 18 fish (Fig. 



122 



R. B. FORWARD, JR., K. W. HORCH, AND T. H. WATERMAN 




-60' 



30 20 10 0% 10% 20% 30% 



c 



-30 




30 20 10 0% 10% 20% 30% 



30 



FIGURE 7. Polarotactic responses of Zcnarchoptcnts to imposed linear polarization. (A.) 
data for all six tested r-vector directions (6 WP conditions) superimposed, pauses only, 
n = 348 ; (B.) same as Figure 7A but total counts used instead of just pauses, n = 578 ; 
(C.) same as Figure 7A but counts for WP 90 excluded; WP 0, 30, 60, 120 and 150 
included, n = 295 ; Eighteen fish. Sectors and significant directions similar to those of Figure 
4 but data folded to 180 as described in text. 

7B). There the distribution is somewhat more random, no sectors are significantly 
avoided and only 26 r / ( instead of 31 ^ of the headings are parallel to the plane 
of polarization. Nevertheless the sector is again the only one significantly 
preferred. 

Finally \ve can show that the strong southerly preference of the 90 WP data 
(Fig. 6Bj adds to but is not by any means the only support in the total WP 
data for orientation parallel to the r-vector. Thus subtracting the 90 WP 
headings from the overall WP distribution yields a new plot (Fig. 7C). However, 
its implications are exactly the same as for the selected pause data (Fig. 7A) and 
the total unselected headings (Fig. 7B). Again the only significantly preferred 
sector is that centered at ( Fig. 7C). This may be taken as evidence that r-vector 
direction can at least partially override orientation clues. 

DISCUSSION 

These results for Zenar chapter us demonstrate a time compensated menotaxis, 
a polarotaxis and, possibly, a negative phototaxis. The last is suggested by the 
significant avoidance of the sun's quadrant evident in distributions plotted relative 
to the solar bearing (Fig. 4C. Fig. 5A, B). However, in the absence of controls, 
other explanations of this are possible such as strong positive preference for dif- 
ferent directions which would leave the solar quadrant relatively empty. 

On the other hand the case for a time compensated menotaxis is well sup- 
ported by the marked preference for southerly headings shown by the fish 



VISUAL ORIENTATION BY ZENARCHOPTERUS 123 

(Figs. 4B, 6A, B, C). This must 1>e lime compensated because South was 90 
counterclockwise from the sun on August 28 and 90 clockwise on August 29. 

Preliminary observations indicated that the fishes' orientation was weak or 
absent when clouds obscured the sun even though only 35-40% of the sky was 
covered. This reinforces the conclusion supported by the heading distributions 
plotted relative to the sun's bearing (Figs. 4C, 5 A, B) that the observed response 
is a /'//o/omenotaxis and not behavior mediated by another untested sensory modal- 
ity c.y., geomagnetic or geoelectric fields (McCleave, Rommel and Cathcart, 
1971 ). In fact the relations between the response distributions obtained with the 
various conditions tested indicate that the preferences observed must be primarily 
visually determined. For example the four fish tested at the end of the August 
29 sequence when the sun's bearing was changing rapidly were disoriented relative 
to the first eight fish run with a steady solar azimuth. 

The menotactic directional preference was more precise for the 18 selected 
fish under natural illumination (NF) than in the totals for all conditions; for the 
NF distribution the only significant preference was due South at 180 (Fig. 6A). 
Similarly a preponderant number of headings occurred in the southerly quadrant 
for the 90 WP condition (Fig. 6B). Note, however, that even if these two 
conditions are subtracted from the total counts, the remaining data (WP at 0, 
30, 60, 120, 150) also show highly significant southward preference (Fig. 6C). 

This preferred geographical direction perhaps relates either to the axis of the 
channel ( 150-300) at the collecting site (Fig. 1) or the direction (150 relative 
to North) which leads from the experimental area (E, Fig. 1) to the site of collec- 
tion (H. Fig. 1). Previous underwater tests had also demonstrated menotactic 
preference for the directions approximately parallel to the channel of the fish's 
normal habitat (Waterman and Forward, 1972). More experiments are neces- 
sary to determine the validity of these several correlations. 

It is not clear from the present results whether the fish are using the sun's 
position directly or the related sky polarization patterns to select their preferred 
geographical headings. The fact that Zenarchoptcnts tends to orient parallel 
to imposed {'-vectors different from that in the sun's vertical ( Fig. 7C) shows that 
they can and do orient to the plane of a superimposed polarization pattern. Also 
as noted above increasing the degree and area of overhead polarization having its 
r-vector parallel to that in the sun's vertical was correlated with a reinforced 
southerly orientation preference. This suggests but obviously does not prove 
that sky polarization plays a role in the responses of Zenachopterus to natural 
illumination. 

However, the strong asymmetry of the present data's geographical distributions 
shows that more than the plane of imposed polarization must be involved. The 
c-vector is of course symmetrical through 180 and therefore by itself indicates 
pairs of opposite geographical directions. Perhaps there is an interaction between 
a polarotaxis symmetrical through ISO" and a response dependent on the sun's 
asymmetric position. 

As mentioned above the fish in preliminary experiments showed little or no 
orientation when the sun was obscured by clouds. A similar correlation also 
appeared in our underwater experiments with Zcnarchopterns (Waterman and 
Forward, 1972). This finding may seem rather surprising in terms of the ex- 



124 R. B. FORWARD, JR., K. W. HORCH. AND T. H. WATERMAN 

* 

pectation that polarotaxis functions merely as a supplementary "sun compass" 
when the sun itself is obscured. However, our results may indicate instead or in 
addition that there is a releasing effect of the sun's disc (or the correlated high 
light intensity) on the underlying orientation preferences. Clearly such hypotheses 
must he tested by further experiments. 

The strong polarotactic preference for orientation parallel to the f -vector of 
the downward illumination is unequivocal in these experiments. Essentially the 
same heading pattern appears in the data corrected to superimpose the (-vector 
whether the six pause distributions for different directions of the polarization plane 
are plotted (Fig. 7A) or the total Wl' data (Fig. 7B), or the pauses minus the 
90 counts (Fig. 7C). Hence the observed behavior must be a polarotaxis and 
not just some other overriding response yielding southerly orientation. 

This polarotactic orientation of ZoHirchoptcrus swimming at the water surface 
differs in several ways from the distributions previously observed underwater 
(Waterman and Forward, l l >72). Thus the present response to the c-vector 
appears considerably stronger in the total data than in the six day sequence of 
underwater experiments. The polarotactically preferred sector in the water surface 
experiments comprised 26% of the distribution ( Fig. 7B ) the corresponding sector 
in the underwater case contained only 19%. 

However, in the underwater data selection of fish and using criteria for pauses 
increased the relative counts in the most preferred direction to 27% whereas the 
corresponding sector for the water surface experiments increased relatively less but 
reached 31% for the pauses (Fig. 7A ) . Consequently future experiments should 
most likely make use of this stronger orientation and the obvious practical advan- 
tages of the water surface type of tests. In addition the experimental situation 
at the water-air interface more closely mimics the environmental conditions usually 
experienced by this particular fish. Hence results like the present ones may be 
a better indicator of normal behavior. 

A second difference between these and the previous experiments is that both 
perpendicular and parallel polarotaxis were observed underwater with the former 
being the more pronounced (\Yatennan and Forward. 1972). Only parallel polaro- 
taxis is significantly preferred in the present results. Third, those submarine 
studies showed that strongest polarotaxis occurred to imposed r-vectors differing 
maximally in direction from that of the natural illumination in the sun's vertical 
(Waterman and Forward. 1972). This is not evident in the present data. 

A fourth difference is that no oblique orientation to the ^-vector is observed in 
the water surface results. In the underwater experiments the fish oriented either 
in the 0, 90, 180, 270 quartet of directions or in one of the four oblique alter- 
natives. Such bimodal preference patterns (parallel or perpendicular vs. oblique) 
are well known in both crustaceans and insects; corresponding four-peak prefer- 
ences occur in cephalopods, too (\Yaterman, 1966). However, the responses of 
these other animals are most likely basitaxes evoked under particular experimental 
conditions rather than the freer menotaxes observed in the present experiments. 

( hir results reported above demonstrate both a menotactic azimuth preference 
which is parallel to the direction of naturally occurring polarized light in the 

lith and the sun's vertical as well as a strong polarotaxis parallel to an imposed 
Hie correlation observed between these two types of orientation is 






VISUAL ORIENTATION BY ZENARCHOPTERUS 125 

suggestive evidence that responses to polarized light may be involved in sun 
compass orientation by this fish. 

The authors are grateful to Mabelita M. Campbell and William F. Corell for 
helpful assistance in carrying out and analyzing these experiments. In addition 
we would like to thank Mr. Peter Wilson and especially Mr. Robert Owen of 
I'alau for their generous aid in making the field work practical. 

SUMMARY 

1. Visual orientation of the surface-living hemirhamphid teleost Zenarchopterus 
has been studied with individual fish swimming in an experimental vessel open to 
the air. Measurements of spontaneous heading preferences were made in the after- 
noon and morning respectively of two successive days, during which the sun's 
bearing differed by nearly 180. Fish were tested under natural illumination of 
sun and sky as well as with six different ^-vector directions of imposed linearly 
polarized light. 

2. Data were selected among other things on the criterion that maintenance 
of a given azimuth direction 20 for a 10 sec period counted as an oriented 
response. Comparison with the distributions of the total measurements justifies 
this selection. 

3. Zenarchopterus avoided the azimuth quadrant towards the sun. This sug- 
gests negative phototaxis but other explanations are possible. 

4. A strong southerly heading preference occurred on both days under natural 
illumination by sun and sky. The same marked preference is also evident in the 
with-polarizer data plotted relative to North. This persists in the residual data 
when the counts are subtracted for the N-S imposed c-vector which parallels the 
sky polarization in the sun's vertical. 

5. Such orientation occurred while the sun's bearings were constant ; when solar 
bearings were changing rapidly orientation was less clear or absent. These results 
support a time compensated sun compass orientation. 

6. Responses to imposed polarization patterns show a strong preferential 
orientation parallel to the (--vector. This persists when the N-S imposed ('-vector 
counts (which demonstrate strong southerly preferences parallel to the sky 
polarization in the sun's vertical) are subtracted from the overall data. Compari- 
son with previous underwater experiments on the same species indicates that these 
water surface data yield stronger polarotaxis and may provide better evidence for 
normal behavior. 

7. The correlation of a menotactic azimuth preference parallel to sky polariza- 
tion in the sun's vertical with strong polarotaxis parallel to the ('-vector provided 
by a polarizer suggests that responses to natural polarized light may be involved 
in normal direction finding by Zenarchopterus. 

LITERATURE CITED 

BATSCHELKT, E., 1 ( '(>5. Stiilistici.il Metliods for the Analysis of Problems In Aninml Onenta- 
tion and Certain Hiolouicul l\hytliins. American Institute of Biological Sciences Mono- 
graph, Washington, 57 pp. 



126 R. B. FORWARD, JR., K. W. HORCH, AND T. H. WATERMAN 

BUDDENBROCK, W. VON, 1917. Lichtkompassbeweguiigen bei Insekten, insbesondere den Schmet- 
terlingsraupen. Sitzitnnsherichte der Hcidelbergcr Akadcmic dcr Wissenschajten B, 
8: 1-26. 

FRAENKEL, G. S., AND D. I.. GUNN, 1940. The Orientation of Animals. Clarendon Press, 
Oxford, 352 pp. 

FRISCH, K. VON, 1950. Die Sonne als Kompass im Leben der Bienen. Experientia, 6: 210-221. 

HARDEN JONES, F. R., 1968. Fish Migration. St. Martin's Press, New York, 325 pp. 

HASLER, A. D., 1966. Undcncuter (,'iiidcposts. University of Wisconsin Press, Madison, Wis- 
consin, 155 pp. 

IVANOFF, A., AND T. H. WATERMAN, 1958. Factors, mainly depth and wavelength affecting the 
degree of underwater light polarization. /. Mar. Res., 16: 283-307. 

JANDER, R., 1963a. Insect orientation. Ann. Rer. Entomol., 8: 95-114. 

JANDER, R., 1963b. Grundleistungen der Licht-und Schwereorientierung von Insekten. Z. 
Vcrgl. Physiol.. 47: 381-430. 

JANDER, R., 1970. Fin Ansatz zur modernen Elementarbeschreibung der Orientierungshandlung. 
Z. Tierpsychol., 27 : 771-778. 

JERLOV, N. G., 1968. Optical Oceanography. Elsevier, Amsterdam, 194 pp. 

KRAMER, G., 1950. Orientiertc Zugaktivitat gekafigter Singvogel. Naturwissenschaften, 37 : 
188. 

KUHN, A., 1919. Die Orientierung der Tiere im Raiim. Fischer, Jena, 71 pp. 

LUNDGREN, B., 1971. On the polarization of daylight in the sea. Kflbenhavns Unirersitet. 
Institut for Fysisk Oceaiiograft, Report No. 17: 1-34. 

McCLEAVE, J. D., S. A. ROMMEL, JR. AND C. L. CATHCART, 1971. Weak electric and magnetic 
fields in fish orientation. Ann. New York Acad. Sci., 188 : 270-281. 

SEKERA, Z., 1957. Polarization of skylight. Pages 288-328 in S. Fliigge, Ed., Handbuch der 
Physik, Vol. XLVIII. Springer- Verlag, Berlin. 

WATERMAN, T. H., 1954. Polarization patterns in submarine illumination. Seienee. 120: 
927-932. 

WATERMAN, T. H., 1955. Polarization of scattered sunlight in deep water. Deep Sea Res., 
3 Suppl.: 426-434. 

WATERMAN, T. H., 1966. Specific effects of polarized light on organisms. Pages 155-165 in 
P. L. Altman and D. S. Dittmer, Eds., Environmental Biology. Federation of Amer- 
ican Societies for Experimental Biology, Bethesda, Maryland. 

WATERMAN, T. H., 1972. Visual direction finding by fishes. In S. R. Galler, K. Schmidt- 
Koenig, G. J. Jacobs and R. E. Belleville, Eds., Animal Orientation and Navigation, 
A Symposium. National Aeronautics and Space Administration, Washington, D. C., 
in press. 

WATERMAN, T. H., AND R. B. FORWARD, JR., 1970. Field evidence for polarized light sensitiv- 
ity in the fish Zenarchoptenis. Nature, 288 : 85-87. 

WATERMAN, T. H., AND R. B. FORWARD, JR., 1972. Field demonstration of polarotaxis in the 
fish Zenarclinpterits. J. E.rp. Zool., 180: 33-54. 



Reference: Bloi. ///., 143: 127-139. (August, 1972) 



HORMONAL FACTORS IN THE CNS AND HEMOLYMPH OF 

PUPARIATING FLY LARVAE WHICH ACCELERATE 

PUPARIUM FORMATION AND TANNING 1 

G. FRAENKEL, JAN ZDARKK.- AND P. SIVASUBRAMANIAN 

Department of Entomology, Vni-rcrsity of Illinois, Urbaini, Illinois 

We formerly reported the existence of a hormone, derived from neurosecretory 
brain cells of fly larvae, which accelerates puparium formation (pnpariation) in 
whole larvae or ligated hind parts. Apparently the same effect appeared when 
hemolymph (blood) from pupariating larvae was used in the place of brain extracts 
(Zdarek and Fraenkel, 1969). It was assumed at the time that the active factor 
in the blood was identical with, or directly derived from, the active component in 
the neurosecretion. Onr assay system has been acceleration of tanning of the 
hind parts of larvae ligated after adequate ecdysone has been released to sustain 
pupariation. This critical period has been reached when the region around the 
posterior spiracles turns red. Since the central nervous system (CNS) in such 
ligated larvae is concentrated in the front part, the hind parts are totally paralyzed, 
and the only criterion for pupariation is tanning. From the few injection experi- 
ments with whole larvae it was learned that the accelerating effect applied not only 
to tanning, but the whole gamut of morphogenetic effects during puparium forma- 
tion which precede tanning. 

In a subsequent study of these morphogenetic events during pupariation 
(Zdarek and Fraenkel, 1972) the significance of certain early processes was recog- 
nized. These consist of a gradual slowing down of locomotion, an irreversible re- 
traction of the anterior body segments into the body, and a gradual muscular con- 
traction and cuticular longitudinal shrinkage into the barrel-shaped puparium. 

The present investigation deals with the roles of substances in the brain and 
blood which promote anterior retraction at the beginning of puparium formation, 
and accelerate contraction and tanning. Surprisingly, the effects produced by in- 
jections of brain extracts and blood have turned out as not identical. 

MATERIALS AND METHODS 

All experiments to be described in the following were performed with the 
fleshfly, Sarcophaga biillata Parker, except where stated otherwise. Extracts from 
the CNS or hemolymph were injected into post-critical larvae in the red-spiracle 
stage, i.e., 3-4 hours before the formation of the white puparium. 

Basically, when testing the activity of various preparations we used 3 different 
criteria : 

1 Supported by NSF grants GB 5441X and GB 23422, and NIH grant 5-K6-GM-18.495. 

2 Permanent address : Institute of Entomology, Czechoslovak Academy of Sciences, 
Vinicna 7, Prague. 

127 



128 G. FRAENKEL, J. ZDAREK, AND T. SIVASUBRAMANIAN 

(7.) The retraction effect 

Blood- or brain fractions were injected into red-spiracle larvae, and the time 
was determined until tin. anterior body segments became retracted (Zdarek and 
Fraenkel, 1972). This time was then expressed as a percentage of the cor- 
responding period in controls which had been injected with Ringer. 

(2.) Cessation of locomotion 

In the normal larva, retraction of the anterior end also signals the end of loco- 
motion, since the mouthhooks play an essential part in crawling. However, under 
certain conditions, retraction is delayed, and is preceded by the contraction and 
shrinkage processes. The latter, then, make locomotion impossible. 

(J.) Acceleration of tanning 

Here, the time between injection into red-spiracle larvae of blood or brain 
extracts and the onset of tanning (darkening) was determined, and expressed as a 
percentage of the corresponding period in controls injected with Ringer. 

In a variation of this test, the procedure, extensively used in our earlier work 
(Zdarek and Fraenkel, 1 () <> ( >), was follo\\ cd. Injections were made into hind parts 
of red-spiracle larvae immediately after they had been ligated. As in our publica- 
tion, the results were expressed as the quotient P/A between the time from injec- 
tion to tanning in the posterior (P), and that in the uninjected anterior part (A ). 

The advantage of using intact larvae in the tanning test lies in the fact that 
anterior retraction and tanning can be determined in one and the same specimen. 
Complications arise when tanning is accelerated to the extent that it starts before, 
or simultaneously with anterior retraction. The advantage of using ligated hind 
parts is mainly the much greater ease and accuracy with which the beginning of 
tanning can be determined in the immobile ligated preparation, as against the 
mobile whole larva. The disadvantage is, of course, the impossibility of deter- 
mining anterior retraction in the same preparation. 

The brain-somatic ganglia complex which in fly larvae is concentrated in a 
single mass in the anterior part of the body w r as dissected from larvae or white or 
orange puparium stages, ground in water in a Potter-Elvehjem homogenizer, 
centrifuged. and injected into larvae or ligated hind parts of the red-spiracle stage, 
as described before (Zdarek and Fraenkel, 1969). Hemolymph was drawn from 
larvae or prepupae of different ages by puncturing with a very fine pipette, and was 
usually pooled from about 20 specimens before injection. 

Brain extracts were injected in equivalents of between 1 and 4 brains per host 
larva, dissolved in :> /xg Ringer solution or distilled water. Hemolymph was in- 
jected in 4, 5, or 10 p.\ per host. 

All figures in the tallies or in Figure 1 represent mean values from 10 to 15 
individual specimens. 

EXPERIMENTS 
(1.) The effect of CNS-extracts 

Table I gives the results of an experiment in which the various processes which 
occur during puparium formation were observed in larvae injected with CNS- 



HORMONES HASTENING FLY PUPARIATION 



129 



homogenates (2 CNS/larva) from red-spiracle larvae, or with hemolymph from 
approximately 1-hour old puparia (4 ^I/larva), and compared with Ringer-injected 
controls. In this experiment each single individual was under observation from 
injection until onset of tanning. 

The CNS-injected larvae started to contract after about 20 minutes and the 
contraction proceeded until the white puparium was completed about 40 minutes 
later. At first they were still crawling in a semi-contracted state, but they had 
virtually come to a stop by 37 minutes. The mouth hooks became withdrawn 
about 10 minutes later, shortly before the white puparium was completed. Tanning 
started soon afterwards. Each of these events took place in only a fraction of the 
time of that in the controls (10.5 to 32% for the different events), but the se- 

TABLE I 

Sarcophaga bullata. The effect of the injection into red-spiracle larvae of homogenates from the 

CNS of red-spiracle larvae, or of hemolymph from the 1-hour puparium stage, 

on the manifestation of various events during puparium formation. 

Time is in minutes after injection. 





n 


Become 
immobile* 


Mouthhook 
withdrawn 


Gradually 
contracting 


White 
puparium 


Tanning 

starts 


Controls (Ringer injected) 


9 












Average time 




159 


185 


190 


200 


234 


Range 




120-240 


125-260 


130-265 


135-270 


165-360 


2 CNS/larva 


8 












Average time 




37 


47 


20 


60 


75 


Range 




20-60 


35-65 


15-25 


45-75 


50-120 


% of controls 




23.3 


25.4 


10.5 


30 


32 


Hemolymph (4 /^I/larva) 


9 












Average time 




16.1 


20.5 


25 


30 


85 


Range 




15-20 


20-25 


20-30 


25-40 


65-130 


% of controls 




10.1 


11.1 


13.2 


15 


36.3 



* Controls and hemolymph injected: Immobilization caused by anterior retraction. CNS: 
Immobilization caused by beginning contraction. 

quence was different under the two circumstances. In the controls, as in normal 
larvae, locomotion ceases with the withdrawal of the mouthhooks, and the contrac- 
tion into the puparium follows later. 

In another experiment with 2 CNS/larva, the effect of CNS -homogenates 
from larvae or prepupae of different ages was compared. On this occasion the 
criteria were termination of locomotion and onset of tanning in unligated speci- 
mens, and tanning in ligated specimens (Table II). It appears that the activities 
of extracts in relation to these criteria differed relatively little in the CNS of dif- 
ferent developmental stages, from the mature larva to 24 hours after pupariation. 
Irrespective of whether the observed differences in the figures are significant, it 
is clear that the CNS retains a considerable activity in all the developmental stages 
investigated. 

Essentially the same results ensued from a similar set of tests where only one 
CNS/host was used (Table II). Here the activities of the extracts apparently 
had decreased at 24 hours after pupariation in tests with unligated larvae, but no 



130 



G. FRAENKEL, J. ZDAREK, AND P. SIVASUBRAMANIAN 



such decrease had occurred in tanning tests with ligated larvae. Extracts from 
only 1 CNS were as active as extracts from two. 

When the equivalents of 4 CNS were injected into each larva, contraction into 
the puparium proceeded even faster and the larvae seemed to be overcome by 
tanning before they had managed to retract the anterior ends. The mouthhooks in 
such puparia either remained outside, or could be pushed outside by squeezing the 
newly formed puparium. 

TABLE II 

Sarcophaga bullata. The effect of the injection of homogenates from the central nervous system, 

taken from larvae or prepupae of different ages, into red-spiracle larvae, on the onset of 

immobilization and tanning during puparium formation. These effects are 

expressed as a percentage of the time after injection in which these 

events occur in the ringer-injected controls 



2 CNS/host 


1 CNS/host 






Tanning 






Tanning 




Immobil- 






Immobil- 




Donor 


ization 
%of 
control 


Unligated 
% 
Control 


Ligated 
P/AU 


Donor 


ization 
%of 
control 


Unligated 

% 
Control 


Ligated 
P/A + 


Mature larva 


46 


34 


0.47 


Mature larva 


39 


31 


0.44 


Early red-spiracles* 


39 


28 


0.46 


Red-spiracles*** 


37 


33 


0.56 


Late red-sp. 1.** 


47 


27 


0.37 










0-hrf 


38 


27 


0.51 


2-hr.f 


32 


32 


0.47 


4-hrf 


43 


30 


0.43 










8-hrf 


45 


37 


0.40 


14-hr.f 


36 


39 


0.60 


16-hrf 


38 


45 


0.49 










24-hrf 


62 


50 


0.50 


24-hr.j 


51 


46 


0.60 



* 3-4 hours before white puparium. 

* 1-2 hours before white puparium. 
*** 2-3 hours before white puparium. 

f After white puparium. 



Period between injection and onset of tanning in posterior (P) and anterior (A) part. 
^ Control 1.67. 
+ Control 1.52. 



In another experiment, CNS's from younger fully grown larvae with the crop 
still full were tested, and there was no difference in activity between them and 
those from red-spiracles larvae. 

(2.) The hemolymph 

Injection of hemolymph from orange puparium stage (about 1 hour after the 
white puparium stage) led to an almost abrupt cessation of locomotion after 15 
to 20 minutes. This period was in the experiment of Table I only 10.1% of the 
time in the Ringer injected controls. In other experiments, injection of hemo- 
lymph produced immobilization in 18, 19, 20, 27 and 28%, respectively, of the 
time in the controls. Such differences were most probably due to differences in the 
late of development of red-spiracles larvae, and in temperature. 



HORMONES HASTENING FLY PUPARIATION 



131 



This immobilization was caused by the irreversible retraction of the anterior 
segments, and was followed by the gradual contraction into the puparium, and 
subsequent tanning. All these processes were enormously speeded up, by com- 
parison with the same processes in the controls. The sequence of events after 
injection of blood was the same as in normal larvae or Ringer-injected controls. 
This differs fundamentally from the sequence after injection of CNS, as described 



100 



80 







60 






20 



\. 

\-Tannmg 
L 




Retraction 



1.5 



1.2 



0.9 



0.6 



0.3 



control -4 -2 4hr 8hr I2hr |6hr 

early late white 
red spiracle puparium 

Age of Donors 

FIGURE 1. The accelerating effects on pupariation in larvae of Sarcophaga bullata, caused 
by the injection of hemolymph from larvae in different states during puparium formation. The 
heavy line represents the acceleration of retraction of the anterior end after injection into 
whole red-spiracles larvae. The effect is expressed as a % of time in the controls. The thin 
line represents the acceleration of tanning in ligated hind parts. The effect is expressed as the 
quotient of the period between injection and tanning in the injected posterior (P), and the 
(non-injected) anterior (A) parts. Each point represents an average from 10 larvae. 

above and also presented in Table I, where immobilization signals the beginning 
of contraction, and where anterior retraction occurs either during or after comple- 
tion of the contraction process. 

The white puparium stage is reached much sooner after injection of hemolymph, 
than after that of CNS-homogenates. Tanning, however, starts at about the same 
time in both cases, or, if anything, sooner in the latter case. The time interval 
between injection and onset of tanning was in both cases about one-third that in 
the controls (Table I). 

Injection of active hemolymph into younger larvae which had already emptied 
their crop but showed no sign yet of red spiracles had little or no effect. 



132 G. FRAENKEL, J. ZDAREK, AND P. SIVASUBRAMANIAN 

Figure 1 shows the results of the injection of hemolymph from donors of dif- 
ferent ages. The hemolymph at the early red-spiracles stage, about 4 hours 
before pupariation had little or no effect on retraction or tanning. Towards 
the white puparium stage it reached a peak of activity which was maintained 
for about 4 hours. All tests with hemolymph from orange puparium stage 
fall under the category of high activity. The retraction effect then decreased 
sharply and had about disappeared 14 hours after pupariation. The tanning effect 
also declined but less decisively than the retraction effect, and was still considerable 
at the 16-hours point. This difference in the decline of these two effect shows, as 
we shall see later, that the two activities are not identical. 

TABLE III 

Sarcophaga bullata. The effect of the injection into intact or ligated hind parts of 

red-sf>iracle larvae of different dilutions of hemolymph from the orange 

puparium stage on acceleration of anterior retraction before, 

and tanning after pupariation 



Dilutions of hemolymph 
(10 lA) 


Acceleration of 




Anterior retraction 
whole larvae 
% of controls 


Tanning 
ligated larvae 
P 






A 


Undiluted 


19 


0.76 


1:1 


24 


0.85 


1:2 


29 


0.96 


1:4 


29 


1.31 


1:6 


30 





1:8 


52 


1.30 


Ringer 


100 


1.34 



(J.) Effects of different fractions of blood 

The hemolymph from orange puparia was centrifuged to separate blood cells 
from plasma, and both fractions were injected separately. The blood cells were 
suspended in the original volume of Ringer. All the activity, both as regards 
acceleration of tanning and retraction, resided in the plasma fractions, while the 
blood cells were entirely inactive. 

(4.) Effect of dilution of blood 

Plasma from orange puparia was tested in a series of dilutions, up to 8 times. 
Vith a 4-times dilution the effect on tanning had entirely disappeared, while the 
retraction effect was still strong with even an 8-times dilution (Table III). 

(5.) Specificity of the hemolymph effects 

The hemolymph (plasma) from white puparia of 3 species of flies, Sarcophaga 
S. argyrostoma, and Calliphora erythrocephala was tested in 5". bullata 
and found equally active, both as regards the retraction and tanning effects. 



HORMONES HASTENING FLY PUPARIATION 

((5.) Origin of the active substances in the blood 

We have seen that the hemolymph of fly larvae in the red-spiracle stage a few 
hours before pupariation entirely lacks any activity in regard to the acceleration of 
retraction and tanning, and becomes active only at the time of pupariation. We 
had at first assumed that the principle which accelerates tanning was released into 
the hemolymph as a neurohormone, originating from the median neurosecretory 
cells in the brain (Zdarek and Fraenkel, 1969), and there was no a priori reason 
to doubt that the same applied to the retraction effect which was only recognized 
later. However, the following experiments show that both activities can arise 
in the hemolymph in the entire absence of the CNS. This was the result of tests 
with hemolymph from hind parts of larvae which had been ligated at a stage when 
it lacked such activities. 

TABLE IV 

Sarcophaga bullata and S. argyrostorna. The effect of the injection of hemolymph from 

the orange piiparium stage of intact larvae or orange colored hind parts, into 

intact, or ligated hind parts of red-spiracle larvae, on acceleration of 

anterior retraction before, and tanning after purariation 



Acceleration of 




Anterior retraction 


Tanning 


Donors 


whole larvae 


ligated larvae 


(10 n\ hemolymph) 


% of control 


P 






A 


S. bullata intact 


24 


0.74 


Hind parts (ligated in red-spiracles stage) 


24 


0.76 


5. argyrostoma intact 


25 


0.65 


Hind parts (ligated precritically, 






pupariation induced by ecdysone) 


19 


0.89 


Ringer control (S. bullata) 


100 


1.28 


Ringer control (S. argyrostoma) 


100 


1.68 



The experiments were performed with hind parts of 5*. bullata and 5". argyro- 
stoma which had been prepared in different ways. Those of S. bullata were 
ligated in the red-spiracle stage, and the blood was taken when the hind part had 
reached the orange stage. Those of ^. argyrostoma were from specimens which 
were wet-treated for 5 days, then injected with ecdysone, ligated and transferred 
to the dry. (Wet-treatment inhibits the release of ecdysone, and thus prevents 
pupariation; Ohtaki, Milkman and Williams, 1968; Zdarek and Fraenkel, 1970). 
Blood for injection was taken from these treated hind parts when they had reached 
the orange stage. The hemolymph of these hind parts, which was inactive at the 
time of ligation, and had remained separated from the source of neurohormones 
in the front part, had become active by the time the cuticles had turned yellow 
(Table IV) exactly as in intact specimens (Fig. 1). 

This shows that both these activities, acceleration of retraction and tanning, 
can arise in the blood in the absence of the CNS at the time when tanning starts. 

We have already shown above (Fig. 1) that blood from normal white puparia 
already contains these two activities, and that, therefore, tanning is not a pre- 
requisite for their manifestation. The following experiments again show that the 



134 G. FRAENKEL, J. ZDAREK, AND P. SIVASUBRAMANIAN 

absence of visible tanning in no way interferes with the appearance of the retraction 
factor in isolated abdomens. Injection of a-MDH into red-spiracles larvae does 
not interfere with the formation of the puparium, nor with the first processes of 
stabilization of the cuticle, but inhibits the subsequent processes of visible tanning 
(Zdarek and Fraenkel, 1972). |>-MDH [ (DL)-a-Methyl-a-hydrazino-j8-(3,4 di- 
hydroxyphenyl) propionic acid (Merck, Sharp, and Dohme)] inhibits DOPA de- 
carboxylation, and thus tanning in adult flies and puparia (Seligman, Friedman and 
Fraenkel, 1969)]. This reaction was used in testing for a possible relation between 
tanning and the appearance of the retraction and tanning effects. a-MDH was 
injected into red-spiracle larvae and their blood tested after the puparia had formed. 
Treatment with a-MDH which prevented tanning in no way interfered with the 
appearance of the retraction effect. 

TABLE V 

The effect of various treatments of hemolymph from the orange puparium stage of 
Sarcophaga bullata on anterior retraction before pupariation 

Anterior retraction Description 

Treatment % of control of effect 

Heating at 80, 10 m (filtrate) 100 heat labile 

Dialysis, 24 hours 31 non-dialyzable 

Freezing (16 hours) 26 remains active 

Freeze drying 28 remains active 
ETOH precipitated, supernatant and 

precipitate tested separately 100 activity lost 
Acetone precipitated, supernatant and 

precipitate tested separately 100 activity lost 

50% (NH 4 ) 2 SO 4 precipitated, filtrate 100 activity lost 

precipitate about 40 remains active 

TCA precipitated (filtrate) 72 some activity remains 



The same result ensued in an experiment where red-spiracle larvae were in- 
jected with a-MDH, ligated, and the blood from the hind parts tested at the 
time when they would bave started to tan in the absence of a-MDH. Since no 
puparial contraction occurs in the ligated hind part, and since tanning is inhibited 
by a-MDH, no visible change occurred in these preparations. The retraction factor 
appeared in these hind parts at the same time, and to the same extent as in ligated 
pupariating hind parts which had started to tan. 

(7.) Characteristics of the accelerating factors from blood and CNS 

A first attempt was made to characterize the chemical nature of the retraction 
factor from hemolymph (Table V). Hemolymph collected from orange puparia 
was submitted to a number of treatments, and then tested. The activity was 
entirely destroyed by heating blood at 80 C for 10 minutes. It was stable to 
freezing and freeze-drying and proved non-dialyzable. It was precipitated by 
alcohol or acetone with a total loss of activity. 

After precipitation with 20% trichloroacetic acid, some activity remained in the 
filtrate. Precipitation, with half-saturated ammonium sulfate removed the whole 



HORMONES HASTENING FLY PUPARIATION 135 

activity into the precipitate, from which it could essentially be recovered, after 
redissolving in water and dialyzing. 

Similar preliminary tests were performed on the nature of the tanning accelera- 
tor in homogenates of the CNS, using P/A test. The results are not strictly com- 
parable to those obtained with hemolymph, considering the differences in prepara- 
tions, test procedure and, presumably, relative concentration, but show very simi- 
lar features. The activity from the CNS is relatively stable to heat and freeze 
drying, is non-dialyzable, and precipitates in 10% TCA and a half-saturated 
solution of ammonium sulfate. It is destroyed by treatment w y ith pronase and 
trypsin, but stable to pepsin. 

All these tests suggest that the active components in hemolymph and CNS 
have similar characteristics, and that we are dealing with proteinaceous sub- 
stances which are easily destroyed by the usual denaturation (and hydrolyzation) 
treatments. 

DISCUSSION 

In an earlier publication (Zdarek and Fraenkel, 1969) we had established a 
neurohormonal effect, deriving from the pars intercerebralis of the brain, which 
accelerates puparium formation in whole or ligated fly larvae when applied after 
the critical period of ecdysone release. A similar effect ensued from the injection 
of hemolymph (blood) from pupariating larvae. It was at first assumed that this 
neurohormone was released from the brain into the blood. 

Subsequently a detailed study of the morphogenetic events which comprise the 
act of pupariation was made (Zdarek and Fraenkel, 1972). This revealed several 
distinct processes of which the following are the most relevant for the present dis- 
cussion : (1.) The irreversible retraction of the first 3 anterior segments into the 
body, (2.) A longitudinal contraction of the body muscles, (3.) A longitudinal 
shrinkage in the cuticle resulting in the smooth surface of the puparium, (4.) Tan- 
ning. In the present study a comparison was made between the effects of injections 
of CNS-material and of the hemolymph from larvae or prepupae of different ages, 
on the different processes which occur during pupariation. Any interpretation of 
the many-fold processes which take place between the release of ecdysone and the 
finished puparium will have to reconcile and integrate the following observations: 

(1.) The activity of the CNS changes little, if at all, from the red-spiracle 
larvae through the 16-hours puparium stage. The blood is inactive at the begin- 
ning of the red-spiracles stage and reaches a maximum activity towards the white 
puparium stage. This activity is maintained for several hours and then declines 
and disappears. 

(2.) Similar accelerating effects ensue from the injection of CNS-extracts or 
hemolymph, but they are not identical. CNS-extracts exert their strongest effect 
on puparial contraction and tanning, while the hemolymph at the peak of its activity 
has the strongest effect on anterior retraction. 

(3.) The active substances in the hemolymph which accelerate anterior retrac- 
tion on the one hand, and contraction into the puparium and tanning on the other 
cannot be identical. The retraction activity is far more dilutable than the tanning 
activity. This is not due to a lower threshold for the former because the hemo- 



136 



G. FRAENKEL, J. ZDAREK, AND P. SIVASUBRAMANIAN 



lymph 16 hours after pupariation still shows a considerable tanning, but no retrac- 
tion activity. 

(4.) The 4 distinct rnorphogenetic processes we have mentioned above are acted 
upon by at least two different entities, one applying to the anterior retraction (X r ), 
and the other to the remaining group of processes comprising the formation of the 
puparium and tanning (X t ). No evidence has so far come to light which would 
suggest X t to involve more than one substance. 

(5.) The different rnorphogenetic processes which occur during normal puparia- 
tion in a temporally ordered fashion (Zdarek and Fraenkel, 1972) are not chain 
reactions, but can occur and be influenced independently from each other. Both 
X r and X t can arise in the absence of tanning (after a-MDH treatment). Accelera- 
tion of tanning occurs in the ligated hind part in the absence of all the other events. 
Contraction into the puparium can precede anterior retraction (by injection of 
CNS-extracts). 



Central 
Nervous 
System 



0. 

anterior 




ECDYSONE 



Gives rise to the appearance 

and accumulation somewhere 
in tissues of 




neurosecretory 
effects 



neuromuscular 
effects 







contraction 
retraction to puparium 

( not necessary for tanning) 



eventually released into 
the hemolymph as 

X r - causes anterior retraction 

X t causes contraction to 
puparium and tanning 



FIGURE 2. Scheme of the interrelationships between neurosecretory and neuromuscular 
effects from the CNS, ecdysone, and the appearance of the X-f actors in the hemolymph, 
during puparium formation of flies. 

(6.) Both accelerating activities X r and X t can arise in the blood in the 
absence of the CNS (in the hind part ligated after the critical period, or ligated 
before, and injected with ecdysone). 

(7.) The factors in the hemolymph and CNS which we have designated as X r 
and X t are most probably proteins and of large enough size to be nondialyzable. 

The following is an attempt to draw up a hypothetical scheme which accounts 
for all these many-fold and sometimes seemingly contradictory and probably largely 
hormonal relationships (Fig. 2). 

It starts with ecdysone the release of which is activated by a prothoracotropic 

neurohormone from the CNS. Ecdysone then causes the appearance of two protein- 

aceous substances which at first must be bound or contained in some tissue present 

in all parts of the body (possibly epidermis or fatbody), and ultimately are released 

into the hemolymph. One of them controls the retraction of the anterior segments 

X r ), and the other affects the other morphogenetic processes, muscular contrac- 

cuticular shrinkage, tanning, which comprise puparium formation (X t ). The 

release of these two factors into the blood is stimulated or activated or accelerated 



HORMONES HASTENING FLY PUPARIATION 

by a neurohormone from the pars intercerebralis of the brain but occurs spontane- 
ously and at a slower rate in the absence of the CNS. 

This scheme seems to account for all observations described in this paper. In 
the normal red-spiracle larva ecdysone is present, and has already given rise to 
X r and X t in a location other than the hemolymph. Injection of CNS-homogenate 
accelerates the release of X r and X t into the blood, and thus accelerates the dif- 
ferent processes which lead to pupariation. A ligature in the red-spiracle larva 
leads to earlier pupariation in the anterior part than the posterior, because the 
former remains subjected to natural activation by the CNS ; injection of CNS 
material into the posterior part causes it to tan first (Zdarek and Fraenkel, 1969), 
because of the plenteous addition of the neurohormone. Injection of active hemo- 
lymph into normal red-spiracle larvae, or ligated hind parts of such larvae, has the 
same accelerating effect because the injection provides already liberated X r and X t . 

The hind part of post-critically ligated larvae contains ecdysone, and X r and X t 
are already present but not yet released into the blood. The blood is at first 
inactive, but eventually becomes active. The hind part of a pre-critically ligated 
larva is either lacking in ecdysone, or may contain it in subeffective doses (Fraenkel 
and Zdarek, 1970; Zdarek and Fraenkel, 1970). Injection of ecdysone leads to 
tanning because it first gives rise to X r and X t in some tissue (as in the red-spiracles 
larva), with subsequent release into the blood. Thus the blood in the hind part 
becomes active although it had been separated from the CNS at a time when it was 
still inactive. 

This scheme postulates the existence in active blood of two different protein- 
aceous factors, and a fundamental difference in the nature of the accelerating 
agent (s) in the CNS and hemolymph. It is hoped that further work into the 
isolation of these factors will bear out these conclusions. 

A possible alternative to the above scheme is based on the assumption of the 
essential identity of the accelerating factors in the CNS and hemolymph. This 
would imply that a product of neurosecretion is stored in the peripheral nerves or 
at the nerve endings during and even before the critical period, and ultimately 
released as the X-factors into the hemolymph through the action of ecdysone. In- 
jection of brain extracts would accelerate pupariation by putting the X factors into 
the hemolymph sooner than they would appear otherwise. Evidence for the work- 
ing of such a scheme would be demonstration of the accumulation in or disap- 
pearance of neurosecretory material from nerves or nerve endings at appropriate 
times, and that of an identity of purified active substances which have been iso- 
lated from the CNS and hemolymph. The possible existence of such a neuro- 
humoral transfer system in the neuromuscular synapses of insects, including a 
fly larva, can be deduced from the work of Osborne (1964, 1967) and Osborne, 
Finlayson and Rice (1971). Whitten (1963) actually interpreted histological 
changes in the dorsal median nerve of fly larvae at the time of pupariation as the 
movement of neurosecretory granules. These nerves disappear soon afterwards, 
after having fulfilled their presumed function in puparium formation. It is not 
obvious how these findings by Whitten can be applied to the case of the pre- 
critically ligated hind part where injection of ecdysone leads to the appearance of 
the X-factors in the hemolymph. 



138 G. FRAENKEL, J. ZDAREK, AND P. SIVASUBRAMANIAN 

Either scheme postulates the existence of two proteinaceous substances inter- 
posed between ecdysone and its visible effects in puparium formation. If our 
reasoning underlying the scheme drawn in Figure 2 is correct, it should be possible 
to induce pupariation by injecting purified X-f actors into a hind part which was 
ligated before the critical period, in the absence of ecdysone. If the alternative 
explanation holds, injection of active material from the CNS into a like prepara- 
tion could achieve the same effect. "Work on the isolation of active material from 
hemolymph and CNS is now in progress and will, hopefully, make the execution of 
these experiments possible. 

These conclusions shed new light on a postulate made by Ohtaki, Milkman 
and Williams (1968) and subsequently elaborated and verified by us (Zdarek and 
Fraenkel, 1970), according to which the action of ecdysone in the formation of 
the fly puparium implied the gradual accumulation of "covert" effects within a 
target organ, produced by a cumulative effect of subeffective doses. It appears 
that our present results on the formation, accumulation, and release into the 
hemolymph of the X-factors exactly conform to these former observations, and 
indeed give a most satisfactory explanation for them. By this reasoning, the 
covert effects in the former studies might be nothing but the processes, set in motion 
by ecdysone, leading to the appearance in the hemolymph of the X-factors and 
finally to pupariation. 

Puparium formation is, of course, a very special case among insects, but it 
would be surprising if the classical function of ecdysone in molting and metamor- 
phosis would not ultimately also turn out to work through further groups of protein- 
aceous substances. 

After this paper was first submitted for publication a paper by Kambysellis and 
Williams (1971) appeared in the pages of this journal with information highly 
relevant to our own conclusions. Both ecdysone and a "macromolecular" factor 
are required for successful spermatogenesis in a silkworm in vivo or vitro, but 
unlike our case where the proteinaceous X-factors appear in the blood in conse- 
quence of an action of ecdysone and conceivably exert their action in its absence, in 
their case the macromolecular factor is already present in the blood and carried 
to the site of action by ecdysone. In both cases the proteinaceous factors exert the 
ultimate effect. 

SUMMARY 

1. Hemolymph or central nervous system (CNS) homogenates from Sarco- 
phaga bullata larvae in various stages during puparium formation were injected 
into red-spiracle larvae (due to pupariate within a few hours), where they cause 
an acceleration of pupariation. The predominant effect of CNS is on puparial 
contraction and tanning, and that of hemolymph on retraction of the anterior end. 

2. The activity of CNS-preparations changes little from the mature larva 
through the 24-hours puparium stage. The activity in the hemolymph is absent 
up to 4 hours before pupariation, at a peak from the white puparium through 4 
hours later and then declines. Sixteen hours after pupariation the effect on retrac- 
tion has disappeared, while that on contraction and tanning is still considerable 

3. The active substances in CNS and hemolymph which accelerate retraction 
xmtraction/tanning are not identical and have been designated as X r and X t . 

respectively. 



HORMONES HASTENING FLY PUPARIATION 139 

4. Both X r and X t can appear in the blood in the absence of the CNS, viz. in 
the hind part ligated after the critical period, or ligated before that period and in- 
jected with ecdysone. 

5. The X-factors in CNS and hemolymph are of the nature of proteins. They 
are denatured by heat, alcohol, or acetone, precipitable by TCA and (NH 4 ) 2 SO 4 , 
non-dialyzable, and destroyed by trypsin or pronase (tested only for CNS). 

6. These observations fit a scheme whereby ecdysone causes the appearance 
and/or accumulation of the X-factors in some tissue and their ultimate release 
into the hemolymph where they induce pupariation. A product of neurosecretion 
in the CNS accelerates the release. An alternative explanation assumes that the 
X-factors originate as a neurosecretion which is stored in the peripheral axons or 
synapses prior to their release into the hemolymph by the action of ecdysone. 

LITERTURE CITED 

FRAENKEL, G., AND J. ZDAREK, 1970. The evaluation of the "Calllphora test" as an assay for 

ecdysone. Biol. Bull, 139 : 138-150. 
KAMBYSELLIS, M. P., AND C. M. WILLIAMS, 1971. In vitro development of insect tissues. II. 

The role of ecdysone in the spermatogenesis of silkworms. Biol. Bull., 141 : 541-552. 
OHTAKI, T., R. D. MILKMAN AND C. M. WILLIAMS, 1968. Dynamics of ecdysone secretion and 

action in the fleshfly Sarcophaga peregrina. Biol. Bull., 135 : 322-334. 
OSBORNE, M. P., 1964. The structure of the unpaired ventral nerve in the blowfly larva. 

Quart. J. Micro scop. Sci., 105 : 325-329. 
OSBORNE, M. P., 1967. The fine structure of neuromuscular junctions in the segmental muscles 

of the blowfly larva. /. Insect Physiol, 13 : 827-833. 
OSBORNE, M. P., I. H. FINLAYSON AND M. J. RICE, 1971. Neurosecretory endings associated 

with striated muscles in three insects (Schistoccrca, Carausius, and Phormia} and a 

frog. Z. Zellforsch., 166: 391-404. 
SELIGMAN, M., S. FRIEDMAN AND G. FRAENKEL, 1969. Bursicon mediation of tyrosine hydrox- 

ylation during tanning of the adult cuticle of the fly, Sarcophaga buUata. J. Insect 

Physiol., 15: 553-561. 
WHITTEN, J. M., 1963. The dorsal nerves of cyclorrhaphan larvae : giant cells in which 

secretory channels appear at the onset of puparium formation. Quart. J. Microscop. 

Sci., 104:217-225. 

ZDAREK, J., AND G. FRAENKEL, 1969. Correlated effects of ecdysone and neurosecretion in pu- 
parium formation (pupariation) of flies. Proc. Nat. Acad. Sci., 64: 565-572. 
ZDAREK, J., AND G. FRAENKEL, 1970. Overt and covert effects of endogenous and exogenous 

ecdysone in puparium formation in flies. Proc. Nat. Acad. Sci., 67: 331-337. 
ZDAREK, J., AND G. FRAENKEL, 1972. The mechanism of puparium formation in flics. /. E.vp. 

Zoo!., 179: 315-324. 



Reference : Biol. Bull., 143 : 140-149. (August, 1972) 



STUDIES ON THE NATURALLY OCCURRING HEMAGGLUTININ 
IN THE COELOMIC FLUID OF AN ASCIDIAN 

M. T. FUKE AND T. SUGAI 1 

Department of Biology, Facility of Science, University of Kanasawa, 

Kanasawa, Japan 

Recent studies on the immunological response revealed that an early indication 
of the appearance of adaptive immunity was found in one of the oldest types of 
vertebrates, a cyclostome (Good and Papennaster, 1964). In invertebrates, al- 
though their exact mechanisms remain obscure, there are many immunelike phe- 
nomena, for example, hemolysis, hemagglutination, bacteriolysis and bacterio- 
agglutination by coelomic fluid, and the recognition of "self" or "not-self" by 
phagocytic cells (Huff, 1940, Briggs, 1966). The question whether the immune 
systems of vertebrates developed from one of the immunelike phenomena of inverte- 
brates has not yet been solved (Burnct, 1968). 

Although natural hemagglutinin can be found in the coelomic fluid of many 
invertebrates, its chemical and biological properties have been poorly understood 
(Marchalonis and Edelman, 1968; Makay, Jenkin and Rowley, 1969; Acton, 
Bennett, Evans and Schrohenloher, 1969). The discrimination between "self" and 
"not-self" by phagocytes was also observed in many invertebrates (Cameron, 1932; 
Aub, Tieslau and Lankaster, 1963), but its mechanism remains to be analyzed. 

Data presented in this paper indicate the presence of hemagglutinin in the 
coelomic fluid of ascidians, uniquely placed between invertebrates and verte- 
brates. We then discuss its properties in relation to those of vertebrates. 

MATERIALS AND METHODS 

Two species of ascidians, Styela plicata (Lesueur) and Halocynthia hilgcndorfi 
f. rittcri (Oka) were used. They were harvested at Noto Marine Laboratory in 
Ishikawa, Japan. The coelomic fluid was collected by cutting the test and mantle 
without injuring the internal organs. After removing the cells by centrifugation, 
the coelomic fluid was collected and stored at --15 C. 

The coelomic fluid was dialyzed to saline of an appropriate concentration used 
for the erythrocyte preparation from each animal. The red blood cells (RBC) 
from various animals were centrifuged at 450 X g and suspended in veronal buffered 
saline, pH 7.2. The final concentration of RBC was adjusted to 10% (v/v, packed 
cells). The number of RBC of 10% (v/v) per ml from various animals were as 
follows; mice (C 3 H(He)), 1.04 r. 10 9 ; guinea pig, 1.02 X 10 9 ; sheep, 4.07 X 10 9 ; 
rabbit, 8.3 X 10; rat, I.I : 10; Crasslus carassins, 4.23 X 10 8 ; Bufo vulgaris, 
1.03 X 10 8 ; Ahilri.i- ti</ris. 3.15 : 10 s . To 0.3 ml of the 2-fold serially diluted 
coelomic fluid, 0.05 ml of red blood cells was added. After mixing completely, 

1 Present Address ; Department of Biology, Faculty of Science, University of Tohoku, 
Sendai, Japan. 

140 



ASCIDIAN HEMAGGLUTININ 141 

the hemagglutinating plate (Tomiki) was incubated at 37 C for 1 hour and 
the hemagglutination was observed. 

For absorption, packed erythrocyte washed several times with saline was added 
to an equal volume of coelomic fluid which had been previously dialyzed to the 
same saline. After incubation at 37 C for 1 hour with occasional shaking, the 
mixture was centrifuged and the supernatant was used for hemagglutinin test. 
The secondary and tertiary absorptions were also tested. 

Sephadex G-100 or G-200 (Pharmacia) was equilibrated by 0.02 M phosphate 
buffered saline (pH 7.8) . Before filtration, the coelomic fluid was heated at 100 C 
for 20 minutes, centrifuged and then dialyzed to phosphate buffered saline over 
night. For trypsin digestion, the reaction mixture used was as follows: 0.1 ml of 
1-5 mg/ml trypsin (Difco, 1:250), 0.3 ml of dialyzed coelomic fluid, 0.1 ml of 
0.2 M phosphate buffer pH 7.8. The reaction mixture was incubated for 30 
minutes, 2 hours and 4 hours at 37 C. 

Periodate treatment was done as follows : the dialyzed coelomic fluid was incu- 
bated at 25 C for 3 hours with the various concentrations of periodate which 
was adjusted to pH 5.4 with 2 N NaOH. Then the coelomic fluid was again 
dialyzed to saline and its hemagglutinic activity was estimated. 

For the observation of phagocytosis, the coelomic fluid was poured out onto a 
cover glass by cutting the test and the mantle. After 10 minutes, the supernatant 
was discarded and coleomic cells adhering to the glass were used for observation. 
After washing with sea water several times the cells were fixed with glutaraldehyde 
at the final concentration of \%. After the fixation had been carried out for 
about 1 hour, the preparation was washed several times by distilled water and then 
stained with Giemsa solution ( X 10) for 20 minutes. 

For other observations on phagocytosis, the coelomic fluid was poured out onto 
the plastic dish (Falcon) and after 10 minutes, the rabbit erythrocytes (fixed with 
1% glutaraldehyde) were added. Twenty minutes later, the cells were fixed with 
1 % glutaraldehyde. 

RESULTS 
The occurrence of hemagglutinin 

Specificity of hemagglutinin. Rabbit erythrocytes were shown to aggregate 
when they were mixed with ascidians' coelomic fluid, even at a high dilution. The 
titer of hemagglutinin of S. plicata to rabbit erythrocyte was + 2 13 and that of 
H. hilgendorfi was + 2 12 . The erythrocytes of fish, frog, and snake were not 
aggregated by the coelomic fluid of either S. plicata or H. hilgendorfi. The 
hemagglutination seems to be restricted to erythrocytes of mammals, such as mice, 
rats and rabbits. The erythrocytes of sheep and guinea pig, although these are 
mammals, did not aggregate in the presence of ascidians' coelomic fluid. There are 
some variations in the agglutinating activity among species of ascidians. The 
coelomic fluid of H. hilgendorfi aggregated rat erythrocytes (titer: + 2 7 ) but did 
not aggregate that of 5\ plicata. Conversely, the coelomic fluid of S. plicata ag- 
gregated mouse erythrocytes (titer: + 2 4 ) but did not aggregate that of H. 
hilgendorfi. 



142 M. T. FUKE AND T. SUGAI 

The absorption test was carried out to determine whether the hemagglutinin 
adhered to rabbit erythrocytes and disappeared from the supernatant as with mam- 
malian antibodies or not. Two milliliters of the coelomic fluid of S. plicata were 
mixed with an equal volume of packed rabbit erythrocytes (1.8 X 10 10 cells). 
After incubation at 37 C for 1 hour, the mixture was centrifuged and the super- 
natant was used for the hemagglutinin test (first step). The secondary and tertiary 
absorptions were done by the same procedure. After the tertiary absorption, the 
supernatant no longer aggregated rabbit erythrocytes. As described later, the 
coelomic hemagglutinin did not change its activity during an hour of incubation at 
37 C 

The specificity of hemagglutinin was studied by the absorption method (see 
Table I). Tests were performed to determine whether the hemagglutinin for rab- 
bit erythrocyte was identical to those of other animals and whether the hemag- 
glutinin for rat or mouse erythrocyte was identical to that for rabbit erythrocyte. 
Two ml of the coelomic fluid of ,S\ plicata and H. hilgendorfi was mixed with an 
equal volume of packed erythrocytes. The number of packed cells of several 
animals per nil were as follows; rat, 1.3 X 10 10 , mice (C 3 H), 1.1 X 10 10 , rabbit, 
8.3 X 10 9 . After incubation at 37 C for 1 hour, the mixture was centrifuged and 
the supernatant was used for hemagglutinin test (primary dilution, X 2). The 
secondary (dilution, X 4) absorption was done by the same procedure. The ques- 
tions of whether the hemagglutinin of H. hilgendorfi is equally effective for the 
aggregation of rat and rabbit erythrocytes, and of whether the agglutinin against 
mouse erythrocytes is identical to the hemagglutinin to rabbit erythrocytes in S\ 
plicata, are answered in Table I. 

It is clear that the hemagglutinin to rabbit erythrocytes was eliminated through 
the absorption by mouse erythrocytes in S. plicata and the hemagglutinin to rabbit 
erythrocytes of H. hilgendorfi was also eliminated through the absorption by rat 
erythrocytes. Conversely, the absorption by rabbit erythrocytes eliminated the hem- 
agglutinating activity to mouse erythrocytes in 6". plicata and to rat erythrocytes 
in H. hilgendorfi. The hemagglutinin of each species of ascidian is thought to be 
homogeneous. However, the hemagglutinin of the two species of ascidians are not 
the same because the coelomic fluid of 5". plicata could not aggregate rat erythrocytes 
which were aggregated by that of H. hilgendorfi. Moreover, the hemagglutinin of 
5\ plicata to rabbit erythrocytes was not absorbed by rat erythrocytes and also that 
of H. hilgendorfi was not absorbed by mouse erythrocytes. Therefore, it may be 
assumed that the hemagglutinin of each of these two ascidians reacts to a different 
position (hapten) on the surface of the rabbit erythrocytes. 

Chemical properties of hemagglutinin. Some chemical properties of hemag- 
glutinin were studied by using the coelomic fluid of Styela plicata. 

The coelomic fluid dialyzed to saline was incubated at 37 C for 5 hours in the 
buffer solutions of various pHs. After incubation, the reaction mixture was 
centrifuged, and after adjusting pH to 7.2 the supernatant was used for the hem- 
agglutinin test. The stability of the hemagglutinin was very high in the region of 
neutral and alkaline pHs but low in strong acidic conditions (Table II). 

The hemagglutinic activity was not affected by the overnight dialysis to physio- 
logical saline. The coelomic fluid of S. plicata was dialyzed to saline containing 
0.01 M EDTA overnight, and its hemagglutinating activity was tested. The activity 



ASCIDIAN HEMAGGLUTININ 



143 



TABLE I 

Specificity of hemagglutinin. For further details, see text 



Ascidian 


Erythrocyte used for 
absorption 


Titers of hemagglutinin to 


Rabbit 


Rat 


Mouse 


Halocynthia hilgendorfi 


No absorption 
Primary (X2) 
Secondary (X4) 


+ 10 X 2' 
+ 10 X 2 6 


+ 10 X 2< 
+ 10 X 2' 







Rabbit erythrocyte 
Primary (X2) 
Secondary (X4) 


+ 10 X 2 
+ 10 










Rat erythrocyte 
Primary (X2) 
Secondary (X4) 


+ 10 X 2< 
+ 10 X 2 1 


: 







Mouse erythrocyte 
Primary (X2) 
Secondary (X4) 


+ 10 X V 
+ 10 X 26 


+ 10 X 24 
+ 10 X 2* 





Styela plicata 


No absorption 
Primary (X2) 
Secondary (X4) 


+ 10 X 2 8 
+ 10 X2 7 





+ 10 X 2 1 

+ 10 




Rabbit erythrocyte 
Primary (X2J 
Secondary (X4) 


+ 10 X 2 3 
+ 10 










Mouse erythrocyte 
Primary (X2) 
Secondary (X4) 


+ 10 X 2* 
+ 10 X 2 3 


: 







Rat erythrocyte 
Primary (X2) 
Secondary (X4) 


+ 10 X 2 
+ 10 X 2- 





+ 10 X 2 1 
+ 10 



TABLE II 
pH stability of hemagglutinin. For further details see text. 



PH 


Buffer (0.05 M) 


Titers of haemagglutinin 


P H 1.9 


KC1-HC1 buffer 


+2 6 


P H 2.9 


citrate buffer 


+2 8 


P H4.3 


citrate buffer 


+ 2 


pH 5.5 


citrate buffer 


+2 10 


pH 6.0 


citrate buffer 


+ 2" 


pH 7.8 


phosphate buffer 


+ 12 12 


pH 8.6 


borate buffer 


+ 12 12 


pH 9.6 


borate buffer 


+ 12 12 



144 M. T. FUKE AND T. SUGAI 

did not change as a result of this dialysis. Nor did the addition of CaCl 2 (2 HIM) 
or MgCl 2 (2 HIM) change the activity. Nor indeed did the addition of CaCl 2 
(2 HIM) and MgCl, (2 HIM) change its activity. 

The coelomic fluid was incubated at C, 37 C, 75 C and 100 C for 
30 minutes. No change of activity was observed at these temperatures. More- 
over, the coelomic fluid was heated at 140 C for 30 minutes in an autoclave, but 
the hemagglutinic activity was not changed. Therefore it seems that the hem- 
agglutinin is not protein. 

The hemagglutinin was digested by trypsin in order to ascertain whether it 
was protein or not. The coelomic fluid of 6". plicata was incubated with trypsin 
(1:250, Difco) at 37 C for several hours. Then the reaction was stopped by 
boiling at 100 C for 10 minutes. The reaction mixture was centrifuged at 450 X 
g for 15 minutes and the supernatant was used for hemagglutinic activity. No 
change of activity was observed due to these procedures. 

TABLE III 

Effect of periodate. For further details, see text. 



Species 


Periodate 

(M) 


Titers of 
hemagglutinin 


Halocynthia hilgendorfi 



0.04 


+ 10 X 28 




0.02 





Styela plicata 


0.004 

0.04 


+ 10 X 2 5 
+ 10 X 2 




0.02 







0.004 


+ 10 X 2* 



From the evidence previously described, such as its heat stability and trypsin- 
resistant properties the hemagglutinin is considered to be a polysaccharide or muco- 
polysaccharide. To confirm this possibility, periodate treatment was performed be- 
cause it is an agent known to destroy saccharide by oxidation. 

The coelomic fluid was treated with various concentrations of periodate and 
then dialyzed to physiological saline. The reaction mixture is as follows ; 1 ml 
of the coelomic fluid that was dialyzed to saline overnight, 0.3 ml of 0.2 M 
citrate buffer, pH 5.4, 0.2 ml of various concentrated periodate which was adjusted 
to pH .5.4 with 2M NaOH. After incubation at 25 C for 3 hours, the reaction 
mixture was dialyzed to saline overnight to remove periodate. After centrifuga- 
tion hemagglutinic activity of the supernatant was measured. The hemagglutinic 
activity was completely destroyed by 0.02 M periodate as shown in Table III. 
Since the hemagglutinin incubated at these pH conditions without periodate did not 
change its activity at '. ' C, the destruction of activity must be due to oxidation 
of saccharide by the periodate. 

The hemagglutinin was not dialyzable at 5 C overnight. Moreover, when 
ammonium sulfate was added to the coelomic fluid up to 50% saturation level, 
hemagglutinin was found in the precipitate. The precipitate was insoluble in water 
but was soluble in 0.85% NaCl. These properties suggested that it is a high- 



ASCIDIAN HEMAGGLUTININ 



145 



weight molecular substance. The rough molecular weight was estimated by the 
gel-filtration on Sephadex. The coelomic fluid w r as heated at 100 C for 15 
minutes, then centrifuged to remove the precipitate and applied to Sephadex G-100 
column. A typical elution pattern was shown in Figure 1. The same experiment 
was done using the Sephadex G-200 column. As shown in Figure 1, two peaks 
appeared, and hemagglutinic activity was found at both peaks when Sephadex 
G-200 was used. The coelomic fluid seems to contain two molecular species of 
hemagglutinin. Assuming that the hemagglutinin is polysaccharide, the molecular 
weight of the smaller one could lie between 150,000 and SOO.OOO and the larger one 
be over 800,000. 



0.6 



0-5 



0.4 



E 
o 

GO 

C\l 

Q 0.2 

O 



Sephadex-GlOO 




Sephadex-G200 




nl 



1 1 1 e r 
(2 n ) 



2 4 



2 4 
t ube number 



12 14 



FIGURE 1. The elution pattern of the boiled coelomic fluid of .S". plicata through sephadex 
G-100 and G-200 column. The coelomic fluid was boiled at 100 C for 15 minutes. After 
centrifugation, the supernatant < O.D. ,,. = 2.4) was applied to the column. The elution 
buffer was phosphate buffered saline, pH 7.2 and the column size was 1.6 X 30 cm. One tube 
contains 6 ml of fractionated solution. Symbols used are: , optical density at 
280 nifjL ; - - O - - O - -, titers of hemagglutinin, 2". 

Effect of the coelomic fluid on phagocytosis 

The coelomic fluid of ascidians contained the hemagglutinin which was con- 
sidered to be polysaccharide or mucopolysaccharide. It also contained a large 
number of coelomic cells. The fluid was examined to determine whether the 
ascidian's hemagglutinin could enhance phagocytosis of cells as with mammalian 
antibodies, although its chemical nature differed. 

Identification of phagocytes in the coelomic fluid. After fixation and staining 
by Giemsa, the coelomic cells of 5". plicata which have properties of adherence 
to the glass surface were observed. Four cell types of phagocytes were dis- 
tinguished. 



146 



M. T. FUKE AND T. SUGAI 



Fine-granular amoeboid cells were abundant and about 80 r / of coelomic cells 
are in this type. The cytoplasm contains many line granules stained reddish with 
Giemsa which characterized these cells (Fig. 2, a). The size of the cells are of 
5-10 fj.. They show active amoeboid movement and phagocytosis (Fig. 2, e). 




. . 



FIGURE 2. Fhagrcylx cells in the on-limnc fluid cf Siyclu /'//<</.'</, (a) fine granular 
amoeboid cells; (h) granular amoeuuid cells; (c) large basophilic cells; (d) vacuolated cells; 
(e) fine granular amoeboid cells which took in rabbit erythrocytes ; ( f I phagocytosis by gran- 
ular amoeboid cell. The scale line indicates 10 /*. 



The cells tend to aggregate when they come in contact with air. And when a toxic 
dose of dye or erythrocytes was injected into the coelomic cavity, these cells were 
also observed to aggregate in a sheet. 

Granular amoeboid cells form about 15^ of coelomic cells. They are large 
granules which are vitally stained with Xile bine. They showed a very active 



ASCI DI AN* HEMAGGLUTINIK 



147 



amoeboid movement and very elongated forms were frequently observed in a smear 
preparation (Fig. 2, b). They showed phagocytosis (Fig. 2, f). 

Large basophilic cells are difficult to find because they are about \-2 c /c of the 
total number of cells. The cell size was about 10-15 p. The cytoplasm was 
stained with a characteristic grayish blue. One or two vacuoles were usually 
observed. The nucleus was relatively small and round, and had an eccentric 
position (Fig. 2, c). Cells usually do not contain erythroyctes, but phagocytosis 
was occasionally observed. 

Vacuolated cells are thin and elongated and have several vacuoles (Fig. 2, d). 
Phagocytic activity of these cells was very evident. The number of the cells 
varied depending on physiological conditions, for example, the cell number increased 
after starvation. 

Effect of the cocloiiiic fluid on phagocytosis. A test was made to determine 
whether the phagocytosis of the coelomic cells of .V. plicata was affected by tin- 
presence of the coelomic fluid which contained the hemagglutinin. 

TABLE IV 
The Effect of the coelomic fluid on phagocytosis. For further details see text. 



Tht- concentration of 
coelomic fluid 


% of phagocytic cells 


s.e.m. 


Sea water 


49% 


1.8 


\ coelomic fluid + \ s.w. 


50% 


3.2 


\ coelomic fluid + \ s.w. 


48%, 


1.8 


\ coelomic fluid + \ s.\v. 


5()' ( 


3.5 


Coelomic fluid 


49.5', 


1.7 



The number of cells per milliliter of the coelomic fluid was estimated by haemo- 
cytometer. Some variations were observed between the different animals. The 
mean value of ten animals was 7.5 X 10" and the standard error of mean was 2.0. 

One milliliter of coelomic fluid was poured on the dishes ( d == 3.2 cm) and 
after ten minutes setting, the coelomic fluid was pooled and centrifuged at 450 X y. 
The supernatant was diluted with sea water at various concentrations (4 coelomic 
fluid + ^ sea water ; ^ coelomic fluid + f sea water ; i coelomic fluid + i sea 
water) and added to the dishes. Fixed rabbit erythrocytes were added and 
after phagocytosis had proceeded for 20 minutes, the cells were fixed. The per- 
centage of viable cells were determined by staining with 0.04% nigrocine. It 
showed over 95% after setting for 30 minutes. The number of coelomic cells which 
had taken an erythrocyte into their cytoplasm was counted. One ml of the coelomic 
fluid which contained the cells (7.0 X 10 <; ) was poured in to a plastic dish (d = 
3.2 mm ). 0.1 ml of red blood cells ( 1 .4 X 10''') was added. Twenty minutes later, 
cells were fixed and stained by Giemsa. 125 X 125 //,- each of phagocytic cells and 
non-phagocytic cells were counted. There were almost 50 cells in total per 125 X 
125 //,-. The cells were counted seven times. The results are shown in Table IV. 
It seems that the coelomic fluid was not essential for phagocytosis. To determine 
the effect of the coelomic fluid on phagocytosis more decisively, an experiment 
using the anti-coelomic fluid antibody is in progress. 



148 M. T. FUKH AXD T. SUGAI 

However, from microscopical observations, it seemed that the coelomic cells 
aggregated with erythrocytes and with each other more actively in the presence 
of the coelomic fluid tlrm in its absence. It also seemed that the coelomic fluid 
enhanced the adhesion of cells to glass surfaces. 

DISCUSSION 

Present results showed that two ascidians possessed hemagglutinins for several 
mammalian erythrocytes in their coelomic fluids. These hemagglutinins are large 
molecules which can he absorbed bv erythrocytes. These properties are similar to 
those of isohemagglutinin present in mammalian serum. Hut the hemagglutinin 
of ascidians is thought to be polysaccharide or mucopolysaccharide because it is 
very heat stable and destroyed by periodate. 

The chemical properties of the hemagglutinin of ascidians are different from 
those of other invertebrates. Mackav ct a!. (19C> C )) reported recently that the 
hemagglutinin of a cravlish was a protein which enhanced adhesion and phago- 
cvtosis of red cells bv the phagocytic cells. The hemagglutinin of oysters was also 
reported as proteinaceous by Acton ct a/. ( 1969). The hemagglutinin of ascidians 
is similar to plant hemagglutinins rather than those of animals (Aub, ct a!., 
1961 ) . 

Hurnet (19(),S) has recently suggested that such hemagglutinins may be fore- 
runners of vertebrate immunoglobulins. However, the hemagglutinin of ascidians 
is not chemically related to vertebrate immunoglobulins. Moreover, structurally 
the hemagglutinin of ovsters is demonstrably different from mammalian immuno- 
globulin (Acton ct <//., 1969). The body fluids of ascidians possess heat labile 
bacterioagglutinin (unpublished data of the authors). There remains the pos- 
sibility that such a protein in the coelomic fluid (other than hemagglutinin) is the 
ancestral precursor of immunoglobulin. 

The biological functions of the hemagglutinin in ascidians still remain obscure, 
and more detailed experiments are required. The hemagglutinin is not essential 
for phagocytosis by coelomic cells in this experimental system, nor was it found 
that the hemagglutinin activated phagocytosis. However, this does not exclude the 
possibility that the hemagglutinin which became bonded to the cell surface of 
phagocytes could play an important role in the discrimination between self and 
not-self. An experiment using the anti-hemagglutinin is now in progress and 
should clarify the problem. 

SUMMARY 

The occurrence of a natural hemagglutinin in the coelomic fluid of solitary 
ascidians, Styclu plicata and Halocvntliiu hil</cndorfi is reported. The hemag- 
glutinin aggregated some mammalian erythrocytes and was absorbed by them. 
'I he hemagglutinins of the two asciclian species are specifically distinct. 

The hemagglutinin of Stvclu plicuta is a large molecule which is very heat 
si able, resistant to trypsin digestion, but is destroyed by periodate. These data sug- 
gest that the hemagglutinin is polysaccharide or mucopolysaccharide. 

"he hemagglutinin has no apparent opsonic effect, but it seems to play a role 
. merence of cell-to-cell and cell-to-glass surface. 



ASCI DI AX HEMAGGLUTIXIX 



149 



LITERATURE CITED 

ALTON', R. T., J. C. BEX. NET, E. E. EVANS AND R. E. SCHROHENLOHER, 1969. Physical and 
chemical characterization of an oyster haemagglutinin. ./. Hiol. Chan.. 244: 4128-4135. 

\i ]-,, J. C., C. TIESLAU AND A. LANKASTER, 1963. Reactions of normal and tumor cell sur- 
face of enzymes. I. Wheat-germ lipase and associated mucopolysaccharides. Proc. 
Nat. Acad. Sci.. 50: 613-619. 

BRIGGS, J. D., 1966. Immunological responses. Pages 259-283 in M. Rockstein, Ed., Physiol- 
ii;/y <if insccta, rolnnic 3. Academic Press, New York and London. 

BURNET, M., 1968. Evolution of the immune process in vertebrates. Nature. 218: 426-430. 

CAMERON, G. R., 1932. Inflammation in earthworms. /. Putltol. Kactcrin!.. 35: 933-973. 

GOOD, R. A., AND B. W. PAPERMASTER, 1964. Ontogeny and phylogeny of adaptive immunity. 
Pages 1-115 in W. H. Taliaferro and J. H. Humphry, Eds., Adrunccs in Immunology, 
Z'olnine 4. Academic Press, Xew York and London. 

HUFF, C. G., 1940. Immunity in invertebrates. Pliysiol. AYr/Viv, 20: 68-88. 

MARCHALONIS, J. J., AND G. M. EDELMAN, 1969. Isolation and characterization of haemagglu- 
tinin from Liiiinlus polyphcmns. J. Mat. Hinl.. 32: 453-465. 

McKAY, D., C. R. JENKIX AXD D. ROWLEV, 1968. Immunity in the invertebrates. I. Studies 
on the naturally occurring haemagglutinin^ in the fluid from invertebrates. Anst. J. 
/:>/>. liwl. Mcd. Sci., 47: 125-134. 



Reference: Biol. Hull.. 143: 150-lhl. (August, V->72) 



DIMENSIONS AND ULTRASTRUCTURE OF TOADFISH GILLS 

G. M. Kl'CHES AND I. E. GRAY 

Research Unit for Coi/if>aratri'e Animal Respiration, The Unii'crsity, Bristol BS8 1UG, England, 
and Department of Zoology, nuke I'liircrsity, Durham, North Carolina 27706 

During the last 20 years the gill areas of many fish species have been measured 
and a fairly comprehensive impression gained of the overall range among tishe> 
belonging to different groups and having a variety of life habits. It has become 
clear that more detailed knowledge is now required for individual species and 
especially in combination with studies on the ultrastructure of the gills and physi- 
ology of gas exchange. Of the marine fishes whose gill areas have been measured, 
the toadfish (Opsanits tan) is of particular interest because of the relatively low 
value found for its gill area (Gray, 1954). This seems to be correlated with the 
sluggish habits of this species and other aspects of its physiology such as its 
respiratory dependence down to verv low O 2 tension in water (Hall, 1929). This 
paper is concerned with the hue structure of the toadfish gills together with a more 
extended analysis of the data summarized previously (Gray, 1954). 

MATERIALS AND METHODS 

The toadfish ranged in size from 15-800 g; the gills were removed, fixed, and 
measurements made according to the method described by Gray (1954). This 
involved the counting of filaments of the three gill arches, sampling of secondary 
lamellae and measurement of their areas. The fish were obtained from Woods 
Hole, Massachusetts, and Heaufort, North Carolina. No significant difference 
was observed between these two populations. These measurements were analyzed 
for the relationship between body weight and different components of the gill area 
using the method of linear logarithmic transformation as described by Muir and 
Hughes (1969). Regression lines were fitted using the method of least squares 
with Wang and Olivetti computers. Other toadfish gills were fixed at Beaufort 
tor inspection of their secondary lamellae, and for electron microscopy after fixa- 
tion in 2.5% glutaraldehyde in cacodylate buffer ( pH 7.2) with subsequent post- 
fixation in \' ', osmium tetroxide and embedded in Vestopal. The material was 
examined under AKI (>(] and Phillips 200 and 300 electron microscopes. 

RESULTS 
dross Morphology 

Toadfish gills are reduced relative to those of most fish for there are only three 
holobranchs on each side. The filaments are widely-spaced along the gill arches 
and this relative coarseness of the sieve extends to the secondary lamellae as there 
are only 10-13/mm on each side of a gill filament (Gray, 1954). 

detailed analysis of a single specimen (400 g) showed that the average 
>er of secondary lamellae/mm was 11.0, 12.7 and ll/> for the tip. middle and 

150 



TOADFISH GILL AREAS AND STRUCTURE 



151 



base of a filament. The filaments of the posterior hemihranch are longer than the 
anterior filaments for the first and second arches, hut the anterior filaments are 
longer on the third arch ( Fig. 1 ). 

Total (/ill area and body wen/lit. The relationship between total gill area and 
body weight for 58 specimens that had been measured, is plotted on log/log coor- 
dinates in Figure 2. It is clear that the gill area increases with increasing si/.e. 
the regression line obeying the equation 



Log A - 5(>0.7 + 0.79 log 
(i.e., A -- 5(>0.7 \Y" 7:i ) 



W 



Correspondingly the regression line for gill area/g against body weight has a slope 
of - 0.217. A summary of the gill area data divided into 50 g classes is given in 
Table 1. The slope of the regression line relating these average areas to body 
weight is 0.779. 



FILAMENT LENGTH 
mm. 



' 

e 
* 



' ' 



/ <t 

I * 








K -& 



1 



FILAMENT NUMBER 



HK, 1. Graphs to show the length of filaments on the different gill arches of the left 
side of a toadfish (en. 400 g). Solid symbols indicate the lengths of filaments of the anterior 
hemibranches in each case. On average, every 5th filament was measured. 

Components <>j the (/ill area. When the measurements for gill area determina- 
tions are examined, it is clear that the increased area of larger fish is mainly due 
to a greater total number of secondary lamellae (Fig. 3 C). The number of gill 
filaments increases rapidly at body weights up to 50 g, but above that size there 
is relatively little increase in the total number of filaments (X ). Filament length 
increases continuously and consequently the number of secondary lamellae (Fig. 
3 ) . This is apparent when the data are plotted on log/log coordinates showing 
that the regression line for an increase in number of secondary lamellae has a slope 
of 0.42 whereas that for filament number increases as W"- OST . Another important 
factor is the increased area of the secondary lamellae themselves, as indicated below. 

The secondary lamellae are relatively large in the toadfish, and as in other 
species their shape varies according to their position on the gill arches and espe- 



152 



(,. \l. HUGHES AND I. E. GRAY 



GILL AREA (mm 2 ) 

TOADFISH 
10 



10 



10 



GILL AREA(mn|gm) 
1000 



(b) 




500 



200 



100 



SLOPE = 0-79 



TOADFISH 
SLOPE = -0-217 



SLOPE = 0-372 



10 
8 



O m 



H 






10 



100 1000 

BODY WEIGHT gm 



10 



10 



i 3, 



BODY WEIGHT 



10 



FIGURE 2. Relationships between areas of the gill system and body weight plotted on 
log/log coordinates: (a) relationship between total gill area and body weight; (b) gill area/g 
and body weight; (c) the unweighted average area of a secondary lamella and body weight. 

dally mi the gill filament ( Fig. 4). Variations in shape and area of the secondary 
lamellae from different parts of the gill are related to the flows of water and blood 
and their role in gas exchange, about which little is known for toadfish. Secondary 
lamella areas can be plotted out in different ways, indicating their increasing area 
in the direction of water flow ( Fig. 4). Such wavs of summarizing the form of 

TABLE I 

Average weights and gill ureas for 50 g classes of 58 specimens of toadfish. Averages 
for fish within the same order of magnitude are also given 



Wt , class 


Average wt. 


Gill area 


g 


Xo. 


mm- fi*\\ 


mmV'g 


0-50 g 


15 


(1) 


5,236 


349.07 


50-100 


69.5 


(7) 


14,199 


204.3 


100-150 


120.4 


(13) 


26,217 


217.75 


150-200 


182 


(13) 


35,248 


193.67 


200 250 


232 


(4) 


48,737 


210.07 


250-300 


277.6 


(5) 


56,906 


204.99 


300-350 


317.5 


(4) 


52,652 


165.83 


350-400 


374 


(2) 


42,168 


112.75 


400-450 


125 


(3) 


71,529 


168.30 


450-500 


471 


(2) 


56,977 


120.97 


500-550 




(0) 






550-600 


560 


(2) 


69,207 


123.58 


600-650 


620 


(1) 


86,867 


140.11 


650-750 




(0) 






750-800 


776 


(1) 


160,362 


206.65 


10-100 


62.7 


(8) 




228.75 


100-1000 


260 


(50) 




191.46 



TOADFISH GILL AREAS AND STRUCTURE 



153 



LLJ 



20 



O 10 

LU 

CO 



I 

o 



5 
100 



10 



10 



TOADFISH 



SLOPE =-0-07 



SLOPE - 0-485 



(h) 



NUMBER OF 
FILAMENTS 



10(X 
600 

200 




(C ) 



TOADFISH 



140 

120 
100 
80 

60 

40 
20 



CO 

[ 



50 



10 



BODY WEIGHT gm 



10 



100 



300 500 700 

BODY WEIGHTgm 



FIGTRK 3. Graphs to show (a) relationship between average number of secondary lamella/ 
mm on one side of a filament and body weight plotted on log/log coordinates; (b) log/log plot 
of the total filament length against body weight and (c) the increase in total number of gill 
filaments and secondary lamellae (clotted line) of 58 toadfish. 



(b) 




1mm 



FIGURE 4. Outline shapes of the secondary lamellae from the tip, middle and base of fila- 
ment 45 of arch 2 of a 400 g toadfish. The change in area of the secondary lamellae in the- 
direction of water flow are plotted (a) as fractional cumulative areas (F) with respect to the 
fractional path length (X/L), (b) as cumulative areas (mnr) along the length of the secondary 
lamella in the direction of water flow. 



154 



G. M. HUGHES AND I. E. GRAY 



TABLE II 

Opsanus tau. Results of regression analysis for the gill area and its component parameters. 





W = 1 g 


\V = 10 g 






Limits 




Limits 






959! Com" 


Tol 




95% Conf 


Tol 


Total fil length (mm) 




363.3 


394.4 




1028 


1150 




S01.2 






923.7 










250.9 


231.4 




830.7 


741.8 


Xo. sec. lam. 'mm on one Mile ol filament 




18.33 


19.53 




14.53 


15.87 


15.86 






13.37 










13.71 


12.87 




12.30 


11.27 


Ave. area of sec. lam. (mm- 1 




0.00781 


(1.1206 




0.1866 


0.2502 




0.0f>037 




0.1412 










0.03726 


O.O<OJ 1 




0.1082 


0.08074 


Total area (mm 2 ) 




921.9 


1 144 




4599 


6024 




560.7 






3459 










341.0 


274.') 


2604 


1929 


\Yt. specific area iinm- yi 




934.2 


1050 


462.9 


551.2 




585.6 






355.4 










367.1 


326.6 




272.9 


220.2 



a secondary lamella are of value in analyses of the O., tension gradients along the 
secondary lamella (Hughes and Hills, l c '71 ; Hughes, 1972a, 1972b). In Gray's 
(1954) original data, secondary lamellae were termed "lamellae" or "platelets" and 
refer to the two secondary lamellae which are more or less opposite at a given 
level on a gill filament. Hence for comparison with data for other species it is 
usually necessary to halve his figures for areas of individual secondary lamellae 
and to double the total numbers of secondary lamellae. 

The increase in total number of secondary lamellae with body weight is largely 
due to the increased length of the gill filaments, for the spacing ( 1/d' ) remains 
relatively constant. The data for total filament length and number of secondary 
lamellae/mm are plotted on log/log coordinates in Figure 3 A and 1> and the rele- 
vant statistical information is given in Table II. Thus: 

Secondary lamellae/mm on one side of filament - ; 15.S(> \Y"" 7t ; and 
Total filament length (mm) := 302.1 W- 485 

l-lne structure oj the f add fish secondary lamella 

The structure of the secondary lamella as seen under the electron microscope 
is similar to that of other teleost fish (Fig. 5) (Hughes and Grimstone, 1965: 
Newsteacl, 1967; Hughes and Wright, 1970; Tovell, Morgan and Hughes, 1970). 
The outer epithelial layers are separated from the pillar cell flanges by a well- 
marked basement membrane which consists of three well-defined layers: (i) an 
outer clear layer, ( ii ) a tine librou.s layer, followed on the inner side by ( iii ) a 
much thicker collagenous layer being about 4 or 5 times the thickness of the other 
two layers, which together constitute the basal lamina (Figs. 6, 8). In some 
places the outer homogeneous layer seems to have protuberances into the inner 
border of the epithelial layers. The collagen fibrils have clearlv defined striations, 



TOAD FISH GILL AREAS AND STRUCTURE 



155 



TABLE 1 1 

Values for 1, 10, 100, tind 1000 g fish are given, together with the confidence and tolerance limit* 



\\ = 100 K 


W = 1000 g 








y _ a \yb 




Limits 




Limits 






95% Conf 


Tol 




95% Conf 


Tol 


b 


s b 


a 


S., 




2920 


343 3 




9191 


10570 










2825 






8638 






0.4854 


0.0174 


302.1 


1.097 




2732 


2325 




8117 


7060 












11.51 


13.12 




9.98 


11.13 










1 1.27 






9.51 






-0.0740 


(1.0137 


15.86 


1.075 




10.98 


9.69 




0.96 


8.13 










0.3624 


0.5529 




0.9301 


1.1329 








0.3347 






0.7879 






0.372 


0.046 


0.0604 


1.274 




0.3090 


0.2026 




0.6676 


0.4672 












23330 


35850 




156500 


225800 










21350 






HI 700 






0.790 


0.047 


560.7 


1.282 




19530 


12720 




1 111 1 )! Ill 


76850 












230.1 


308.0 




152.4 


191.8 










1 15. 7 




130.9 






-0.217 


0.045 


585.6 


1.265 




202.1 


151.0 




1 12.4 


89.32 











repeating every 040 A. The collagen layer is particularly thick next to each pillar 
cell body where it gives rise to the columns. 

Because of its thick collagen layer, the toadfish gill is especially suitable for 
inspection of the structure of the columns which, as in other species, are extra- 
cellular. The number of columns/pillar cell is about five. The not uncommon 
folding observed in the pillar cell columns (Fig. 6) perhaps suggests a contracted 
condition of these cells at fixation. In both transverse and longitudinal sections 
of the pillar cells, it is apparent that the columns also contain a type of fine fibril 
which is not, however, a direct continuation of the fine fibrous layer of the basal 
lamina (Fig. (>). In the pillar cell flanges, fibrils appear in cross-section which 
are very suggestive of collagen. Another interesting feature observed in toadfish 
pillar cells is the presence of cytoplasmic processes jutting into the blood channel 
from the main cell body or its flanged part. These processes sometimes contain 
what appear to be collagenous fibrils. The pillar cell body contains many types 
of granule and is well provided with cytoplasmic filaments suggestive of contractile 
protein, particularly in the neighborhood of the columns. There is also some evi- 
dence of such filaments in the endothelial cells which line the marginal channel of the 
secondary lamella. Typical endothelial granules (Weibel and Palade, 1964; Hughes 
and Wright, 1970; Weibel and Hughes, in preparation) were specially prominent 
in the toadfish and their presence in these cells only and not in the pillar cell flanges 
was very clearly defined in most cases. In addition, the endothelial cells have many 
pinocytotic vesicles and specially large ones are often seen bordering the collagen 
layer of the basement membrane. Unlike the comparable layer of the mammalian 
lung, this endothelium has no underlying basal lamina. 

The outer epithelial layers have a number of points of interest. Microvilli are 
not very obvious in most sections but there seems to be some surface sculpturing 
as there are deep invaginations between epithelial cells, especially in the crypts 



156 



G. M. HUCHKS AND I. K. CRAY 



. 




^J^^MSS 




l : j(a/i<K 5. Opsanus tint. K'lL-rtmn micrograph t<> ^hou tin- IUIMC .structure of a secondarj 
laim-llu cut transverse to the direction of blood flow. Tliree pillar cells (PC) are visible 
separating and lining the blood channels. Notice the sculpturing of the outer epithelial layer 
( Kpi ) and the different types of lymphocyte ( monocytes and macrophages ) to be found in the 
lymphoid space ( Ly Sp ) . betueen the tuo epitlielial layers. The basement membrane (BM) 
sejiarates the inner epithelial layer ( F.p-_. ) irom the pillar cells. 



TOADFISH GILL AREAS AND STRUCTCRE 157 

between secondary lamellae. Many fine-folded junctions are found between cells 
of the outer epithelial layer and where these come to the surface they sometimes 
resemble microvilli similar to those noticed in the pollack between adjacent epi- 
thelial cells. Desmosomes are also visible at these junctions. Epithelial cells in 
the region outside the marginal channel are often particularly thick (Fig. 7). 
Frequently the outer layer of epithelial cells seems to form a continuous coat which 
may be separate to some extent from the underlying epithelial cells. These "lym- 
phoid" spaces often contain a number of lymphocytes of different types but the 
space clearly does not contain plasma. Transitional stages from monocytes to 
macrophages are often visible (Fig. 5). Amoebocytes are also present in the 
blood channels (Fig. 7), but no connection has been established between these 
channels and the intra-epithelial spaces. The outer surface of the epithelial cells 
is more darkly staining than the rest. Chloride cells are common, both in the 
crypts and other regions of the secondary lamella and often seem to be separated 
from the outer surface by the outer epithelial layer. The cytoplasm of the chloride 
cells is also somewhat unusual. 

The water/blood pathway is relatively thick in certain parts of the sections, 
but in others it may be as thin as 3 //m. The epithelial layers usually constitute 
from 4 to ;/ of the total water/blood distance. The flange and endothelial layers 
are very thin and. as mentioned above, the basement membrane and particularly 
its collagen layer, is noticeably thickened in this species. The collagen layer is at 
least 1 /tin thick. 

DISCUSSION 

The toadfish data analy/ed in this paper are more extensive than that available 
so far for adults of any other marine species. The results obtained are in sub- 
stantial agreement with those given for tunas ( Muir and Hughes, 1969) with which 
they contrast considerably because of the great differences in activity of the two 
species. Some of the most obvious differences in gill dimensions are shown in 
Table III which summarizes the "a" and "b" values in the relationship Y = a\V b , 
where Y is the gill area or its constituent parameters. Clearly the slope of the 
log/log regression line for total gill area of tunas is greater than that of toadfish 
and similar differences are seen in the component parameters; for both fish they 
are all of the same order of magnitude. Far greater differences are observed in 
the "a" or intercept values, <'.</-. that for total area of tuna is 6 times that of toadfish. 
This is made up of a 1 0-fold difference in the figure for total filament length and 
a four-times greater number of secondary lamellae/mm. However, for the area 
of an average secondary lamella the "a" value for toadfish is about 14 times greater 
than for tuna. These differences clearly support the generalixation that more 
active fish tend to have a greater number of closely-spaced secondary lamellae 
which are of relatively smaller area (Hughes, I960). Figures for the dolphin 

FIGURE 6. A single pillar cell from a secondary lamella of the toadfish showing its flange 
( PC Fl ) lining the blood spaces, the thickened collagen layer ( Col ) of the basement mem- 
brane, and a single column ( Cmn ) in longitudinal section. The fine fibrous (FF) and outer 
clear ( Cl arrow) layers of the basal lamina can easily be distinguished. A transverse section 
across a single column is shown at higher magnification as an insert, bottom left. 



158 



(i. M. HUGHES AND I. E. GRAY 




FIGURES 7-8. 



TOADFISH GILL AREAS AND STRUCTURE 



159 



TA.BLK 1 1 1 

Comparison of the intercept (a) and slope (b) of the regression lines for the 
components of the gill areas of toadfish, Coryphaena and tunas* 





Toadfish 


Coryphaena 


Tunas 


a 


t. 


a 


b 


a 


b 


Total filament length (mm) I,** 
Secondary lamellae/mm 1 
on one side of filament ft 


302.1 
15.96 


0.485 
-0.079 


1879 
33.81 


0.431 
-0.036 


5594 
60.87 


0.382 
-0.089 


Average area of sec. lam. (mm-) bl 
Total area (mm 2 ) A 


0.0604 

560.7 


0.372 
0.79 


0.0377 
5208 


0.327 
0.713 


0.0046 
3151 


0.583 
0.875 



* Based on Muir and Hughes (1969). 
** Symbols as in Hughes (1966). 



fish, Coryphaena (Table HI ) are closer to tuna, and data for other fish fall between 
the toadfish and Cor\phacna. 

From the respiratory point of view, a very important parameter is the distance 
( t ) separating the blood and water which is substantially greater in the toadfish 
than in the tuna. The general relationship between (X consumption (Vo 2 ) an d 
area of the respiratory surfaces (A) is given in the equation: 



V , = 



KA AP,,. 



which mav be rearranged : 



Vo 2 

Al' 



= K 



0., 



t= D " 



the diffusing capacity of the tissue barrier of the gills (Hughes, 19721)). K is the 
permeation constant of Krogh expressed as ml O 2 ///,rn/cm 2 /mrn Hg and is usually 
assumed to be the same for the different layers of the water/blood barrier which 
has an overall thickness t. However, if there are marked differences in K between 
these layers then the much greater thickness of the collagen layer in toadfish could 
be significant. 

The available figures give estimates for gill area of a typical 100 g toadfish of 
about 21,000 mm- whereas that of a bltiefin tuna of the same size would be of the 
order of 200,000 mm-. The water/blood distances are approximately 5 p\\\ for 

FIGURE 7. Section through the marginal channel of a toadfish secondary lamella. The 
nucleus (End N) of an endothelial cell is clearly visible but the section only passes through 
the edge of a pillar cell of the outer row. Bordering the latter a greatly thickened collagen 
layer (Col) can be seen. The epithelial layers along the outer edge of the marginal channel 
(M Ch) are thickened and separated by lymphoid spaces (Ly Sp), so that the water/blood 
distance is much greater here than on the lateral aspects of this channel. 

FIGURE 8. Higher magnification electronmicrograph of a part of the marginal channel 
(M Ch) to show the different components of the water/blood barrier. The section passes 
through a flattened endothelial nucleus (End N) and typical granules (Gr) are visible in this 
cell which clearly differentiate its cytoplasm from that of the pillar cell flanges (PC Fl) which 
line most of the blood channels. 



160 <;. M. HUGHES AND I. E. GRAY 

toadfish and 0.5 //.m in tunas ( Hughes, 1970). Consequently the diffusing capaci- 
ties ( D t ) are about 42 K and 4000 K, respectively. Thus the same differences in 
Oo tension across the gills would result in the transfer of an amount of O L , that is 
about 100 times greater for the tuna than a toadfish. This example clearly empha- 
sizes the importance of the differences in area and thickness of the two species, 
but as yet no physiological measurements have been made of the diffusing capacity 
of the gills (IX) in these two species so that these estimates must remain ana- 
tomically-based. 

Ultrastructural studies of the toadfish gill have also served to emphasize one 
or two interesting features which are probably general for most fish gills. 

As mentioned previously, the collagen layer of the basement membrane is par- 
ticularly well developed in this hsh and emphasizes its importance as a supporting 
structure in these relatively coarse gills, which may continue to function when the 
fish is out of water. Two epithelial layers are present as in most other species, 
but in the toad fish the space observed between these two layers is particularly 
noticeable. The presence of macrophages and other leucocytes in the lymphoid 
space suggests a protective function, perhaps analogous to the alveolar macrophage 
of the mammalian lung. Clearly the respiratory surface of the fish being con- 
stantlv ventilated by water could not have macrophages on its outer surface. 
Thus the presence of such cells between the two epithelial layers can be related to 
the difference in respiratory medium. This space has generally been omitted in 
discussions of the water/blood barrier of fish. If it is taken into account, the con- 
stituent layers can be listed as follows (1) outer epithelial layer; (2) lymphoid 
space; (3) inner epithelial layer; (4) basal lamina; (5) collagen layer; (6) pillar 
cell Mange instead of epithelium (1, 2, 3), basement membrane (4 and 5) and 
pillar cell flange (6 ). 

Comparison of the line structure of the toadfish secondary lamella with that 
of tuna is also instructive and emphasizes the correlation between structure of the 
respirator}- surface and the habits of the animals. 



We wish to thank Knut Schmidt-Xielsen and Vance Tucker for providing and 
assisting with computer facilities. Analysis of this data was begun during a visit 
of G. M. H. to the Department of Zoology at Duke University, N. Carolina, and 
both there and at Beaufort he enjoyed excellent hospitality. 

SUMMARY 

1. This analysis of the measurement from the gills of about 60 toadfish using 
log/log transformation has shown that the gill area and its constituent parts in- 
creases with body weight as follows : 

Total area (mm-) " 560.7 \V- 79 

Total filament length (mm) = 302.1 W- 485 

Secondary lamellae/mm on one side of filament = 15.99 \V-"-" 7r> 

Average bilateral area of a secondary lamella ( mm- ) = 0.06 W- 372 

2. A comparison with the corresponding data for other fish, particularly tunas, 
shows differences in the values of these relationships and confirms the general 



TOADFISH GILL AREAS AND STRUCTURE 161 

conclusion that the gills of more sluggish fish have a smaller number of larger 
secondary lamellae and relatively wider spaces through which the water flows. 

3. An electron microscope study of the toadfish secondary lamella has shown 
the same basic structure as in other teleost fish but the collagen layer of the base- 
ment membrane is especially thick. Also noticeable are distinct lymphoid spaces 
between the two epithelial layers and the presence of stages in the development of 
cells concerned with the protection of these layers. The water/blood barrier there- 
fore comprises the following layers in certain regions: (1 ) outer epithelial layer, 
( 2 ) lymphoid spaces, ( 3 ) inner epithelial layer, ( 4 ) basal lamina, composed of 
outer clear and inner fine fibrous layers, (5) collagen layer, (6) pillar cell flange. 

LITERATURE CITED 

GRAY, I. E., 1954. Comparative study of the gill areas of marine fishes. Hiol. Bull., 107: 
219-225. 

HALL, F. G., 1929. The influence of varying oxygen tensions upon the rate of oxygen con- 
sumption in marine fishes. Amcr. J. Pliysiol.. 88: 212-218. 

HUGHES, G. M., 1906. The dimensions of fish gills in relation to their function. /. /:.r/>. 
Bio!.. 45 : 177-295. 

HUGHES, G. M., 1970. Morphological measurements on the gills of fishes in relation to their 
respiratory function. Folia Morphologica, 18: 78-93. 

HUGHES, G. M., 1972a. Distribution of oxygen tension in blood and water along the secon- 
dary lamella of icefish gills. J. E.vp. Bio/.. 56: 481-492. 

HUGHES, G. M., 1972b. Morphometrics of fish gills. Rcspir. Physiol.. 14: 1-26. 

HUGHES, G. M., AND A. V. GRIM STONE, 1965. The fine structure of the secondary lamellae 
of the gills of Gadus pollachiits. (Juart. J. Microscop. Sci., 106: 343-353. 

HUGHES, G. M., AND B. A. HILLS, 1971. Oxygen tension distribution in water and blood at 
the secondary lamella of the dogfish gill. /. E.r/>. liiol., 55 : 399-408. 

HUGHES, G. M., AND D. E. WRIGHT, 1970. A comparative study of the ultrastructure of tin- 
water/blood pathway in the secondary lamellae of teleost and elasmobranch fishes 
benthic forms. Z. Zellforsch. Mikrosk. Anat., 104 : 478-493. 

MUIR, B. S., AND G. M. HUGHES, 1969. Gill dimensions for three species of tunny. /. E.vf>. 
fii,'l.. 51 : 271-285. 

NEWSTEAU, J. D., 1967. Fine structure of respiratory lamellae of teleostean gills. Z. Zell- 
forsch. Mikrusk. Anat. } 79: 396-428. 

TOVKLL, P. W., MIRIAM MORGAN AND G. M. HUGHES, 1970. Ultrastructure of trout gills. 
17tli Coin/ri's Intermit, dc Microscopic Rlcctroniquc, Grenoble, 3: 601. 

WEIBEL, E. R., AND G. E. PALADE, 1964. Xew cytoplasmic components in arterial endothelia. 
./. Cell AW., 23: 1 ( '1-212. 



Reference: Biol. Hull., 143: 162-174. (August, 1972) 



A HIERARCHY OF HISTO-INCOMPATIBILITY IN 
HYDRACTINIA ECHINATA l 

FRANCES B. IVKER - 

Zoologv Dcpiti'tiiii'iit. Indhiiui University und .Marine Biological Laboratory, 

H'o(>t1s Hole. Massachusetts 

There is a striking consistency in the biochemistry and ultrastructural morphol- 
ogy of all living cells. These cells do, however, recognize differences among them- 
selves and react accordingly. Cellular recognition mechanisms are operative in 
dissociated embryonic cells derived from different organisms (chick and mouse), 
so that cells of like function remain together in chimeric aggregates, while those 
derived from different organs segregate (from each other) ( Moscona, 1957). 
Dissociated sponge cells segregate according to species (Humphreys, 1963), and 
cells from different tissues of the same organism segregate from each other within 
the initial reaggregate mass (Steinberg, 1962a, 1962b, 1963). Mechanistic ex- 
planations of cellular segregation focus on differences in cellular adhesiveness 
( Townes and Holtfreter, 1955) due to stereospecificity of binding sites; species 
specific extracellular binding molecules (Humphreys, 1963); or variations in 
thermodynamic energies of adhesion (Steinberg, 1962a); or specific recognition 
sites on cell membranes for specific histocompatibility antigens (Burnet, 1970). 

Recognition and interaction exist between unlike cells as well. Endocrine 
secretions affect specific target organs. All inductive processes involve molecular 
mediators. Normal development is a well integrated temporal series of inductive 
interactions in which one tissue chemically initiates change in a second tissue. 
All differentiation and morphogenesis is the result of delicately balanced intra- and 
and intercellular stimulation and feedback control systems. Occasionally there is 
a breakdown in the system, resulting in hyperplastic or neoplastic growth. 

The study of developmental deviations in simple organisms may reveal the 
mechanisms of similar imbalances in more complex species. The Coelenterata 
offer a simple system in which to approach these developmental mechanisms. 

The main focus of this study is the "overgrowth" phenomenon, a hyperplastic 
development of stolons resulting from a histo-incompatibility among genetically 
different strains of Hydmctinia ccliiuata isolated from nature. Some attention will 
also be given to some aspects of normal development which have been misinter- 
preted in the literature. 

MATERIALS AND METHODS 

Hydr actinia echinata is an encrusting, colonial marine hydroid usually found on 
gastropod shells that have been appropriated by the hermit crab Pat/urns. All 

1 This investigation was supported in part by National Institutes of Health Grant GM 11555 
and a grant from the Indiana University Foundation. 

- Author's current address : Department of Biological Sciences, Louisiana State Uni- 
versity in New Orleans, New Orleans, Louisiana 70122. 

162 



HYDROID HISTO-COMPATIBILITY 163 

strains used in this study were isolated from shells collected in the Woods Hole, 
Massachusetts area. 

A hierarchy of overgrowth potential was established twice, using two sets of 
ten strains each. The first group of animals (Group 1 ; 1-5 male, 6-10 female) 
was started from animals scraped from the surface of shells collected in August 
at the time when the colonies were in full sexuality. The second group (Group 
1 1 ; 1-5 male, 6-100 female ) was collected in December when the colonies showed 
juvenile or regressive sexual development. Clones derived from the mating of 
these colonies were designated by the number of both parents (i.e., colony 8 
mated to colony 4 produced 8 I- offspring). In the 1 generation, the overgrowers 
were designated 8-4(0) and their overgrown siblings were labelled S-4(X). 

Stock strains were started by scraping a piece of mat from a colony growing 
on a hermit crab shell. One or two feeding hydranths were then teased from this 
mat and their proximal, cut ends held in close contact with a glass slide by a 
single loop of thread tied around the slide. Within 24 hours in the faster growing 
strains, stolons grew out of the cut ends, adhered to the glass and thereby held 
the feeding hydranths to the slide. The thread was then removed. Three to five 
colonies were started under each thread. Stolons grew out from the transplanted 
hydranths so that they eventually met stolons of the other transplants. If the 
colonies were of the same stock, they fused, forming a continuous colony across 
the width of the slide. If the transplants were of different stocks, hyperplastic 
stolons were produced by one or both of the colonies. 

The first group of experimental animals was raised on glass slides, held vertically 
in glass staining racks in standard (2V' X 3" X 3") staining dishes. The dishes 
were placed on a slow, gentle horizontal shaker. The second group of animals 
was grown on slides in staining racks that were placed in a plexiglass tank 3" X 
15" X 3". Water was circulated by a vibropump and passed through glass wool 
and charcoal filters. 

The hydroids were kept in natural sea water that was pasteurized by heating to 
80 C on two consecutive days and aerated for 30 minutes before use. The water 
\vns changed every 7 days. Temperatures ranged between 19 and 22 C during 
the course of the study. The cultures were fed freshly hatched brine shrimp 
(Artemia salina ) once a day. 

Precautions were taken to keep the cultures as "clean" as possible, but due to 
their living food and their initial isolation directly from nature, the cultures were 
occasionally infected by bacteria, algae and ciliated protozoa. The contaminants 
were kept in check by periodic light swabbing of the slides and colonies with cotton 
wound on a thin glass rod. Heavy infestations of ciliates were treated by placing 
the cultures in a dilute ( 100 units ml ) solution of Mycostatin for 20 minutes, 
which cleared the slide of surface ciliates without apparent harm to the hydroids. 
Heavily infected areas of the colonial mat were cut away and the excised tissue 
was quickly replaced by healthy new tissue. 

In order to test strain compatibility, two strains were placed on a slide and 
allowed to grow until the stolons contacted each other. All possible binary combina- 
tions (45) of the ten strains in Group I and Group II were tested. Compatibility 
was also tested on gastropod shells to insure that the overgrowth phenomenon ob- 
tained in nature as well as in the laboratorv. Hermit crabs were removed from 



164 FRANCES B. IVKER 

their shells and kept in isolation. Hydranths of the strains to be tested were 
tied in place. When the colonies appeared fastened to the surface by stolonic 
growth, the threads were cut and the crabs were allowed back into their shells. 
Each crab was kept in its individual finger bowl to avoid abrasive contact with 
other crabs. 

Vital staining was accomplished by feeding stained Artcnua nauplii to the colony 
to be dyed. One drop of either 0.1 r < aqueous Nile Blue Sulfate or Neutral Red 
was added to 30 ml of sea water. Freshly hatched Artcinla was left in this stain- 
ing medium for 24 hours. They were then picked in a hand-held micro-pipette and 
presented within the tentacle range of the colony to be stained: red to one colony 
and blue to the other colonv of a binary combination. Saturation feeding once a 
day for (v-10 days produced enough color in the growing stolons to determine 
the origin of solons in an overgrowth tangle and to detect any exchange of ma- 
terial between the colonies. Reciprocal staining procedures were initiated in several 
incompatible pairs to negate the possibility of chemical involvement of the dye in 
hyperplastic stolon development. When normal feeding was resumed, the vital 
dyes faded in about two weeks without apparent harm to either colony. 

Controlled breeding was accomplished In isolating sexually mature female 
colonies at least two days before mating. A slide containing a male colony was 
introduced into the staining dish, facing the remale colony at a distance of 1-2 cm. 
Eggs were observed on the bottom of the dish the next morning and elongated 
planula larvae 12-18 hours later. If larvae were allowed to remain scattered on 
the bottom of the breeding dish, metamorphosis into small four-tentacled, feeding 
hydranths was observed in 10-40',/ of the larva in 7-21 days. Larvae were also 
picked up in a micro-pipette and introduced onto the clean shell of a hermit crab, 
resulting in a higher rate of metamorphosis (30-60%). When a new colony 
\vas well established, either on glass or shell, one or more feeding hydranths of 
normal size were transferred to a glass slide, as described earlier, and a stock 
colony established. The conditions responsible for the induction of sexuality and 
metamorphosis are highly unpredictable at this time. Most breeding was done at 
Woods Hole in July and August, but crosses have been made in Indiana in De- 
cember, and Crowell (1Q50) and Hauenschild (1954) regularly raised sexually 
mature colonies in mid-winter at Tubingen. Germany. 

RESULTS 

Normal i/nra'tli 

Within twentv-four hours after placing a newly isolated hydranth in contact 
with a slide, stolons grew out along the glass surface, firmly attaching the hydranth 
to the substratum. Stolons grew in all directions, branching and anastomosing 
freely within the two dimensions of the slide. They displayed no gravitational 
tropisms and always maintained contact with the substratum. Other hydranths 
sprang up along the stolons, increasing the feeding and growth potential of the 
new colony. The area between the branching and anastomosing stolons was sub- 
sequently filled with tissue, histologically described (Berrill, 1953; Bunting, 18 C >4 ) 
as an ectodermal mat penetrated by interconnected, endodermally lined gastro- 
vascular channels. 



HYDROID HISTO-COMPATIBILITY 165 

The rate and pattern of stolon growth and mat formation varied among the 
different strains being raised under identical culture conditions. This fact was also 
noted by Schijfsma (1939). The same growth patterns appeared in all colonies 
derived from the same original colony isolated from nature. 

Alterations in the culture conditions brought about changes in these develop- 
mental patterns. Lack of water movement, reduced aeration and infrequent water 
changes led to a retardation of stolonic outgrowth, but not to a concomitant 
reduction in the rate of hydranth production. The result was a smaller colony 
with a greater density of nutritive zooids. Return of the colony to standard 
culture conditions produced a renewed stolonic outgrowth similar to that seen in 
control colonies of the same strain. Cleaning the slide surface and cutting awav 
infected perisarc restored normal fusibility. 

(irowth on the surface of a gastropod shell was slower than on glass, and mat 
formation followed more closely behind stolon growth. In shells occupied by a 
crab, spines were produced after three or four weeks, but on empty shells no spines 
were produced, although the colony grew well and even reached sexual maturity in 
some crises. 

()rcr</roi\.'th ( hyperplastic slulmis ) 

In order to test compatibility, colonies of two strains were started on a slide, 
as described earlier. Each developed normally, sending branching, anastomosing 
stolons in all directions, always maintaining contact with the glass surface, until 
stolons of one colony made contact with stolons of the other colony. At this point, 
one of the colonies started to produce abnormal stolons; they rose up off the slide, 
losing contact with the substratum, and formed hyperplastic, tangled masses. They 
did not immediately fuse with other stolons of the same colony as they normally 
would have done when growing Hat on the glass surface. During all the tangling, 
they maintained the general direction of growth toward, and over the other colony. 
The hyperplastic stolons grew over the stolons, mat and feeding hydranths of the 
other colony, cutting off its contact with the food supply and eventually causing 
its death. 

Closer observation of the overgrowth phenomenon indicated that physical con- 
tact of incompatible strains was essential to the induction of hyperplastic growth, 
and only those stolons in contact with the overgrown colony were affected. Al- 
though material was seen to circulate throughout a colony via the gastrovascular 
system, hyperplastic stolons were not observed in other areas of the overgrowing 
colony. Attempts were made to induce the production of stolonic overgrowth 
throughout a colony by immersing it in a crude brei made from another colony 
known to induce overgrowth by the contact method, but no positive results were 
obtained. 

Although no abnormal growth patterns were observed in other areas of a 
colony involved in overgrowth production in the contact stolons, it was noted that 
normal growth was quantitatively reduced, while the rate of growth of stolons 
actively involved in overgrowth was increased. The colony as a total unit ap- 
peared to be concentrating its corporate nutritive resources in the production of 
the stolonic tangle at the expense of normal growth in other areas. It was also 



166 FRANCES B. IVKER 

observed that colonies actively involved in the overgrowth process showed delayed 
sexual maturation, as compared to control colonies of the same strain. 

A tangled mass of stolons could reach a height of 5 mm above the surface of a 
slide and extend 25 mm across an overgrown colony. When these hyperplastic 
stolons completely covered the underlying colony and reached the glass surface on 
the far side, they immediately returned to their normal, two-dimensional, anasto- 
mosing growth pattern, regardless of hyperplastic growth still in progress in lateral 
areas not yet completely covered. 

The overgrown colony did not die immediately. First, the feeding hydranths 
were reabsorbed in the area initially covered by the stolonic tangle. As the over- 
growth progressed, more and more nutritive zooids were reabsorbed until all were 
gone, leaving the overgrown colony with no means of obtaining food. There were, 
however, large food reserves within the mat, and if the overgrowth was stripped 
away before too much of these reserves had been utilized, the overgrown colony 
could again produce feeding hydranths and resume normal growth with no obvious 
ill effects. If the tangle remained, the tissue of the overgrown mat slowly utilized 
its food reserves and died within 3-6 weeks. During this time, the stolonic tangle 
above remained static, while normal growth and hydranth production continued 
on either side of the tangled mass. Anastomosis of the upper surface stolons of 
the tangle was observed at about the same time we assume death occurred in the 
underlying tissue. Observation of cross sections of the mass, using the dissecting 
microscope, revealed a spongy center of empty perisarc that formerly contained 
stolons of the overgrowth. The surface was a continuous mat of living ectoderm 
containing numerous channels lined by endoderm. These channels became clearly 
denned when vital stain was applied via the food source. 

Spontaneously, over the entire irregular surface of the mass, feeding hydranths 
appeared, with the same density and morphology as those seen in areas of normal 
growth. If the colony had shown retarded sexual development, gonozooids ap- 
peared, evenly distributed on the flat, normal mat and on the surface above the 
tangle. 

No transfer of colored material could be detected between incompatible colonies 
that had been vitally dyed in contrasting colors, even as the overgrown colony 
diminished in volume, no absorption of colored material was noted in the over- 
growing colony. 

Colonies derived from the same source retain their compatibility even after 
long periods of separation. Fusion was observed between colonies derived from 
two older colonies of strain #8, Group II. which had overgrown strain #2 and 
strain #7 respectively, and had been isolated as individual colonies for ten months. 
Their temporal separation and physiological activity in the overgrowth process 
had not interfered with or altered their compatibility ( fusibility ) . In similar tests, 
hydranths from the top of a tangled mass were explanted to a slide between 
explants of the two strains whose interaction had given rise to the tangled mass 
of stolons. In all nine cases, the tangle explant colony fused with the colony 
that had produced the overgrowth and it, in turn, produced hyperplastic stolons 
when contact was made with the overgrown strain, again indicating no alteration 
in tissue compability as a result of participation in the overgrowth process. 

In rare instances, both colonies produced abnormal stolons upon contact, but 
one eventually outproduced and overgrew the other. There were situations in 



HVDKOII) HISTO-COMPATIBILITY 



167 



which related, hut not genetically identical colonies, such as parent and offspring, 
or two strains with one common parent, contacted each other ; each produced a 
limited number of abnormal stolons, which were quickly replaced by an abnormal, 
thickened area of mat tissue on both sides of the line of contact. Vital staining 
showed that there was no fusion or transfer of material between the two colonies, 
but neither was there any hint of overgrowth by either one or the other. 

Hierarchy 

In selecting ten colonies at random from nature and setting up all ( 4r> ) pos- 
sible binary combinations, it was observed that if colony A overgrew colony B, 
and if colony B overgrew C, it could be predicted that colony A would overgrow 
colony C. There was a definite, predictable hierarchy of overgrowth, the strongest 

TABLE I 
Rt'sitlls at himiry combination Group I 



SI lain 


Sex 


Overgrows 


Is overgrown by 


Rank in hierarchy 


2 


& 


1 3 45 6 8 9 10 


7 


1 


5 


cf 


3 4 67 8 9 10 


2 


2 


3 


& 


1 4 67 8 9 10 


2 5 


3 


8 


9 


1 4 67 9 10 


2 3 5 


4 


7 


9 


1 J 


f 46 9 10 


3 5 8 


5 


1 


<? 


4 6 9 10 


2378 


6 


9 


9 


4 6 10 


123578 


7 


4 


<? 


6 10 


1235789 


8 


Id 


9 


6 




12345789 


9 


6 


9 







1 2 3 4 5 7 8 9 10 


10 



-f- Oare not in expected positions. 



overgrower being listed as #1 in the hierarchy. The strain that was overgrown by 
all the others was designated as tenth in the hierarchy. The other strains all 
fell in order, depending on their relative frequency as an overgrower. 

In the first set of data presented in Table I, there are two discrepancies in the 
hierarchy. Strain #7 was scored as overgrowing #2, and #1 was scored as 
overgrowing #5, which is contrary to expectations based on data derived from the 
other 43 binary combinations in this group. Neither of these results could be 
checked due to an accidental loss of all Group I strains. There was one apparent 
discrepancy in the predicted results of Group II. In the first trial, #7 appeared 
to overgrow #X, but subsequent tests of the pair, both on slides and crab shells, 
accompanied by vital staining, proved #8 to be the overgrower as would be pre- 
dicted from other tests, leading to the possibility that there may have been a 
labeling reversal in the initial trial. 

Although two strains proved incompatible upon stolonic contact, this did not 
affect sexual interaction. It was, therefore, possible to produce second generation 
strains. There were five matings in Group I and three in Group II. 



168 



FRANCES B. IVKER 



The larvae were allowed to metamorphose on the hottom of the breeding- 
dish. As growth continued it was observed that stolonic compatibility and incom- 
patibility existed between siblings and that either fusion or overgrowth occurred 
at the junction of any two colonies. By transferring colonies to slides and setting 
up from nine to twenty-four possible combinations per mating, it was determined 
that only two classes of offspring existed: the overgrowers (i.e., 7-4(0)) and the 
overgrown (7-4(x)). All those that overgrew their siblings were compatible, 
and all those that were being overgrown were fusible with each other. Because 
of the large numbers of larvae and limited space, no accurate determination of 
the percentage of offspring in each class was made. 

TABLE II 
Results of binary combinations Group II 



Strain 


Sex 


Overgrow? 


Is overgrown by 


Rank in hierarchy* 


10 


9 


1 


i 


3 


4 5 


6789 











1 


8 


9 


1 


2 


3 


4 5 


679 


10 








2 


9 


9 


1 


> 


3 


4 5 


6 7 


8 


10 






3 


4 


c? 


1 


~> 


3 


5 6 


7 


8 


9 







4 


1 


cf 


2 


3 


5 


6 7 




4 


8 


9 10 




5 


1 


9 


2 


3 


5 


6 




1 


4 


8 9 10 




6 


6 


9 


2 


3 


5 






1 


4 


789 


10 


7 


5 


c? 


2 


3 








1 


4 


678 


9 10 


8 


3 


cf 


2 










1 


4 


5 6 7 


8 9 10 


9 


2 


tf 













1 


3 


456 


7 8 9 10 


10 



DISCUSSION 

The initial work on Hydractinia was basically descriptive of normal develop- 
ment (Bunting. 1894; Teissier, 1929; Teissier and Teissier, 1927; Schijfsma, 
1935, 1939; Berrill, 1953). Teissier (1929) and Schijfsma (1939) noted that 
there was fusion into a single colony when stolons derived from different planula 
larvae made contact, but an anomaly was noted by Schijfsma in 1939 (page 101 ) : 
"It looks as if the growing borders of two colonies, in striking together and check- 
ing each others progress, are stimulated to very active growth and ramifications ; 
resulting in the formation of a dense fringe of intertwined stolons." 

Schijfsma vaguely speculated about a "timing factor'" but noted that this 
"fringe" did not appear when a colony met itself on the other side of a shell. This 
indicated that the "fringe" was not a normal marginal phenomenon. Toth (1967) 
enlarged upon this suggestion, calling it "temporal specificity" (page 131), and 
claiming that compatibility, even of clonal colonies was variable with time. He 
stated that all colonies were compatible early in life, but became increasingly 
selective; eventually colonies of the same strain could not fuse. In this study, 
however, incompatibility was demonstrated between newly metamorphosed (2-3 
day) colonies by the production of hyperplastic stolons which overgrew sibling 
colonies ; while continued compatibility was demonstrated by the fusion of explain 
colonies derived from two colonies of the same strain that had been established as 
individual colonies ten months earlier. Both colonies had undergone the physio- 



HVDROII) HISTO-COMPATIBILITY 169 

logical stresses of overgrowth production and still retained their compatibility. 
Colonies derived from hydranths on the surface of the tangle fused with clonal 
colonies of the strain that had originally produced the tangle, indicating that even 
stolons that initially appeared unable to fuse during the overgrowth process retained 
their histo-compatibility when they returned to normal colonial metabolism. 

Another point of variance in Toth's paper ( 1967) was his report that colonies 
on glass slides usually reached a maximum diameter of 5-10 mm before the "en- 
dogenous limit of closed periderm is attained" (page 131). He does, however, 
mention later that no such limit is seen in nature. No such limits were seen in 
the present study. A "dirty" slide whose surface is covered by a layer of bacteria, 
algae and/or protozoa will inhibit or halt free stolon growth. The vulnerable 
stolon, with its high surface-to-cell-mass ratio, is poisoned or damaged by these 
other organisms faster than it can regenerate new tissue, and eventually new stolon 
growth stops. The mat may slowly expand for a while longer under these condi- 
tions, but this too eventually ceases. Toth's (1967) description of a "limiting 
periderm" and reduced stolon growth can be explained as an artifact of substandard 
culture methods. His report of incompatibility (lack of fusion) between colonies 
of the same strain may be due to a build-up of necrotic tissue or bacteria-encrusted 
perisarc at the contiguous margins of the two colonies, preventing perisarc disso- 
lution by ectodermal enzymes or preventing cell-to-cell contact and tissue fusion. 

Toth (1966, 1967) reported free stolon growth in \() ( / ( of his strains. Both 
Hauenschild and Kanellis (1953) and Toth (1967) suggest this may be due to 
poor nutrition. The current study of well fed and well aerated stocks produced an 
open stolon pattern in 80^ of the strains tested, with a varying stolon/mat ratio 
characteristic of each strain and reliably reproduced by all colonies derived from 
that strain. Reduced oxygen supplies in the medium retarded stolon growth. The 
mat continued to grow as a slowly expanding circle from the point of implantation. 
A return to more advantageous culture conditions brought a renewed outgrowth 
of freely anastomosing stolons. 

Crowell (1950), Hauenschild ( l c >54), and Toth (1967) discuss lack of fusion 
between strains and regard this as incompatibility, but none of these investigators 
records or discusses the induction of hyperplastic stolons. M tiller (1964), how- 
ever, does report the formation of stolonic "knots" to which both colonies con- 
tributed stolons. 

The role that particular strain plays in relation to any other strain, either as 
overgrower or overgrown colony, is not a chance occurrence. Among the strains 
tested, a very definite hierarchy emerged in both sets of experiments. 

There appeared to be a correlation between growth potentials, as related to 
colony morphology, and the position of a strain in the hierarchy. A fast-growing, 
highly stolonic strain is likely to rank higher on the scale of overgrowth potential 
than a slower-growing, short-stolon, large-mat former ; the correlation is not 
absolute, however, and rank by growth rate becomes difficult to determine among 
strains of similar developmental morphology. 

Vital staining experiments showed that in some cases both colonies initially 
produced abnormal stolons. Miiller (1964) reported the participation of both 
colonies in the formation of a stolonic "knot." In almost all cases, one strain was 
superior in hyperplastic stolon production and the other began a regression that 



170 FRANCES B. IVKER 

ended in death. Mviller suggests that this regression is caused by a toxin produced 
by the overgrowing colony. Stripping away the overgrowth leads to rapid ( 2-4 
days) and full recovery, suggesting that the regression is the result of mechanical 
stresses applied by the hyperplastic stolons. When 80^ of the colony being 
overgrown is covered, the other 20% spontaneously withdraws the feeding hy- 
dranths in the uncovered area. It is suggested that this is the result of a general 
physiological regression of the entire colony caused by an unfavorable balance 
between metabolic requirements and nutritional acquisition rather than a reaction 
to a specific toxin. 

The term "hierarchy" suggests the work of Steinberg (1 ( >(o) and his hier- 
archy of embryonic tissue associations and segregations in tissue culture. His 
results indicated a predictable position in a cellular reaggregate. 1 're-cartilage had 
the highest probability for interior position, liver the highest probability for the 
outside, with heart-cells variable, based on the particular binary combination. 
Steinberg repeated these experiment.-, with several embryonic tissues establishing a 
hierarchy of potential position at the center of the mass. The explanation offered 
by Steinberg involved an "energy of adhesion" between cells, so that if two cells 
of type A displayed a significantly higher attraction for each other than did those 
of type B or an A cell for a B cell type, the A type cells would tend to aggregate 
together with as much mutual surface contact as possible, thereby excluding cells 
of the B type from their midst and forcing them toward the periphery of the 
reaggregate cell mass. 

"Energy of adhesion" is a cell surface phenomenon in which cells seek the 
"lowest energy state" or most stable adhesive condition possible. The adhesive 
mechanism could, in principle, be a quantitative one based on the number of ad- 
hesive sites available or a qualitative one based on the specificity of the various sites. 

Applying these hypotheses to the hierarchy in Hydractinia, two possible mech- 
anisms may be proposed, both involving a surface-hound molecule produced bv 
the ectoderm. The first hypothesis involves a quantitative variation in this sub- 
stance ; the second suggests a qualitative difference. When stolons of the same 
strain meet, the quantity and/or quality of the molecule is identical and fusion 
results. If, on the other hand, there is a significant difference in either quantity 
or quality of the substance, the stolons recognize this difference and react by the 
production of hyperplastic stolons by one or both colonies. 

Looking first at the qualitative hypothesis, we can postulate a mechanism similar 
to serotypes found in I'aniiiicciinn. ( Sonneborn. 1948). Incompatible strains 
would produce strain-specific proteins which could induce hyperplasia in other 
strains. The intensity of the reaction could be due to the degree of difference 
in the surface molecule. It was noted that the intensity of incompatibility, as 
indicated by the speed and quantity of induced hyperplastic growth, varied con- 
siderably among the strains tested. Certain slow-growing strains, such as 4. (> 
and 10, Group I and strains 2 and 3, Group II, induced a much weaker reaction 
from the #1 strain in their respective hierarchies than did other overgrown strains 
higher up in rank. 

The maximum degree of difference that will trigger the reaction is limited, 
however, as evidenced by the fact that contact with other hydroids (i.e., Cainpa- 
niilaria. Bougainvillia and Podocoryne) failed to elicit a response. This indicated 
that the overgrowth reaction was not a simple antigen-antibody-like response to a 



HYDROID HISTO-COMPATIBILITY 1/1 

foreign protein, but rather a highly specific, intra-species selective mechanism that 
plays a role in genetic distribution within the H \dractinla population. 

Considering the quantitative difference hypothesis, the colony with the higher 
concentration of the particular molecule in any pair might he the inducer. This 
would explain a strain's shift from overgrower to one which is overgrown as a 
shift in the relative amount of this surface molecule, when compared to the quantity 
of this substance in the other strain of any particular combination. This would 
be similar to Steinberg's hypothesis of differential adhesion based on a quantitative 
difference in available binding sites. The hierarchy then would be a quantitative 
ranking of the presence ( or absence ) of this inducer molecule. 

Preliminary experiments ( Ivker. 1967 ) with reaggregation of dissociated, 
stained endoderm cells do not demonstrate histo-incompatibility between strains 
on the cellular level. 

Braverman, M. (Allegheny General Hospital, Pittsburgh. Pa. ) has photographic 
and histologic evidence that even in normal stolon fusion, in the related encrusting 
species Podocoryne, the advancing stolon tip produces a substance that causes an 
increase in the size of the epidermal cells that the tip is about to contact. An in- 
crease in epidermal cell size is also seen in the overgrowth stolons. Miiller 
(1904) mentions a hyperplasia of epidermal cells in both the stolons and the 
mat in the area of contact between incompatible strains. 

It is suggested that each strain produces a substance ( in greater quantity at 
the growing tip) which has a hyperplastic effect on epidermal cells. In the con- 
tact of stolons of the >ame colony, or colonies of the same strain, the stolon tip 
is thought to produce an en/yme which dissolves the perisarc in a small area and 
facilitates cell-to-cell contact and fusion of the gastrovascular cavities. When strains 
are incompatible, the surface substance on the growing stolon again induces a 
reaction in the epidermal cells in the area of contact. These cells produce more of 
their own surface substance which in turn induces hyperplastic growth in the on- 
coming stolons. This accelerated growth rate may prevent the accumulation of 
sufficient enzyme at the stolon tip, thereby preventing the fusion of actively grow- 
ing hyperplastic stolons with each other. Miiller ( 1904 ) proposes a similar mech- 
anism (page 241) when he ascribes "wild" stolon growth to a stimulation of 
dormant developmental potential in one strain by a foreign (incompatible) strain. 

Attempts to characterize the inducing substance have given rise to several 
hypotheses. It is either bound to the ectodermal surface or it slowly diffuses 
through the perisarc from the ectoderm. The failure of an incompatible strain 
to "condition" the medium in which it was grown, and the failure of a crude brei 
of incompatible colony to induce hyperplastic stolon development may be evidence 
of the small quantities produced by any given colony and/or the failure of the 
material to reach a concentration above the threshold required for hyperplastic 
induction. 

As was noted earlier, stolonic contact between incompatible strains was essential 
for the induction of hyperplastic stolons. Only growing stolons were affected 
(i.e., those laid down before contact was made remained unaffected). Nothing 
was carried via the gastrovascular system to induce abnormal stolon development 
in any area of the colony not in direct contact with the overgrown colony, leading 
to the hypothesis that the overgrown colony acts as an inducer. The inducing 
agent is either surface bound or a large molecule that cannot easily diffuse through 



1/2 FRANCES B. IVKER 

tissue or water in sufficient concentration to induce overgrowth in any but con- 
tiguous stolons. There must be continued production of the substances, since the 
stolonic mass bult up, but remained as a deep tangle of stolons as long as there 
was living inducer colonv below it. Stolons that reached the far side of the over- 
grown colony and made contact with clean glass, resumed their normal growth 
pattern and regular hydranth production. Although stolons in the tangle acted as 
connectives between colony mat on either side of the overgrown colony, no induc- 
tive material was transported in either direction to affect hydranth production. 
Hyperplastic growth was localized to that area in direct contact with the incompat- 
ible colony. The only area lacking feeding hydranths and not displaying stolon 
anastomosis and mat formation was the tangle mass itself and this situation was 
temporary. The eventual stolonic fusion, mat formation and appearance of nutri- 
tive polyps was believed to coincide with the death of the overgrown colony and 
the cessation of its production of inducer. It is suggested that the inducer sub- 
stance promotes increased growth ( i.e., hyperplastic stolons that do not anastomose) 
yet it inhibits differentiation of specialized tissue areas like feeding or reproductive 
zooids. Perhaps the increased growth rate prevents the concentration of material 
required for hvdranth formation. 

Although there was no gross morphological change observed in the outlying 
parts of the overgrowing colony, there was, nevertheless, an effect felt throughout 
the colony. The hyperplastic mass resulted from an accelerated deposition of ma- 
terial in one area, which could only be made at the expense of growth in other 
areas. The stolonic tangle increased the mass of the colony, but the nutritive 
capacity of the colony did not undergo a concomitant increase, due to the absence 
of feeding hydranths in the tangle area. There was, therefore, a definite decrease 
in peripheral stolon growth and mat production as compared to control colonies. 
There was not total cessation of normal growth patterns, but a noticeable retarda- 
tion as the colony concentrated its productive energv and nutritional resource in 
the overgrowth mass. 

Although the induction of sexuality is far from understood, optimal nutrional 
conditions are a prerequisite to the process. Sudden starvation caused the trans- 
formation of gonozooids into nutritive polyps. The reverse was not true. Gono- 
zoiids arise dc noi'o from the mat tissue and were not derived from pre-existing 
nutritive structures. It is postulated that the accelerated proliferation of stolon 
tissue that constituted the overgrowth mass depleted the nutritional reserves 
ncessary for gonozooid production, thereby retarding sexual differentiation. 

Sex does not appear to be related to compatibility. Colonies initially deter- 
mined to be 7-5 ( X ) , Group I immediately after metamorphosis, but raised on 
separate slides, subsequently turned out to be of opposite sex. Fusion of these 
colonies after initial growth had established the individuality of each colony could 
produce sexual chimeras in the fusion zone, as reported by Muller and Hauens- 
child. Muller (l ( 'Mi reported having to try many binary combinations (which 
produced "knots") before he found two strains that were even partially compatible. 
In this case, he reported fusion of the endodermal gastrovascular cavities, while 
the mat ectoderm of the female colonv formed large masses that appeared to invade 
the male colony. The masse 1 - eventually withdrew. The separation of apparently 
fused, partially compatible colonies was observed in this study, especially in times 
of physiological depression. In trying to localize the source of incompatibility, it 



HYDROID HISTO-COMI'ATIBILITY 173 

was found that empty perisarc did not elicit a reaction, indicating that the inductive 
substance was not an integral part of the molecular structure of the perisarc. 

The variability of morphology seen between various strains, the consistent-} 
of morphology within a strain and the clear-cut orderliness of the hierarchy indi- 
cate a genetic control mechanism. Hauenschild initially proposed a single locus, 
six-allele system to explain the results of his histo-incompatibility studies, but 
ultimately abandoned the hypothesis when it proved inadequate to deal with the 
complexity of accumulated data. 

Looking to other biological systems for a clue, the colonial tunicate Botrylns 
schlosscri presents a seemingly similar example of histo-incompatibility in which 
the vascular systems of incompatible strains fail to fuse, but no hyperplastic 
growth is observed. Karakashian and Milkman (1967), Milkman (1967) postulate 
a multi-allelic system, but assign no definite number of alleles. 

At the present time, there is no definite evidence to support a multi-allelic 
versus a multi-genie hypothesis, or to rule out a complex combination of the two. 
With the demonstrated feasibility of controlled laboratory mating and the use 
of morphological! v unique strains, it is hoped that some insights will soon be 
gained into the mechanisms of the genetic transmission of incompatibility. 

The ability to produce hyperplastic stolons and a high place on the hierarchy 
appears to have a selective value in nature. It is hoped that morphologic markers 
can soon be found that can help trace the ecological distribution of the genetic 
factors responsible for the overgrowth phenomenon, and that the inductive mech- 
anism can be more firmly established. 

SCM MARY 

1. Hydractinia represents a simple system in which to study induction, cellular- 
recognition mechanisms, and hyperplastic growth. 

2. Among various strains isolated from nature, there is a tissue incompatibility 
upon contact which results in the production of hyperplastic stolons (overgrowth) 
in one or both colonies of any binary combination of strains. The induction of 
hyperplasia probably involves surface-bound molecules produced by the ectoderm. 

o. A hierarchy of hyperplastic potential was established in two groups of ten 
strains each. A correlation between colonial morphology and rank in the hier- 
archv was noted. 

4. Consistency of morphology and intermediate forms of incompatibility be- 
tween related strains (;'.<\, parent-offspring, half-sibs) suggests genetic control of 
histo-incompatibility and hyperplastic growth. 

LITERATURE CITED 

BERRILL, N. J., 1953. Growth and form in gymnohlastic hydroids. VI. Polymorphism within 
the Hydractiniidae. /. Morphi>L, 92 : 241-272. 

BUNTING, M., 1894. The origin of the sex-cells in Hydractinia and Podiicnrvnc and the de- 
velopment of Hydractinia. J. Mor[>hoL. 9: 203-237. 

BURNET, F. M., 1970. A certain symmetry : Histocompatibility antigens compared with immuno- 
cyte receptors. Nature, 226: 123-126. 

CROWELL, S., 1950. Individual specificity in the fusion of hydroid stolons and the relationship 
between stolonic growth and colony growth. Auat. Rcc.. 108: 560-561. 



174 FRANCES B. IVKKR 

HAUENSCHILD, C., AND A. KANELLIS, 1953. Experimentelle Untersuchungen an Kulturen von 
Hvdractinia echinata Flem. zur Frage der Sexualitat und Stockdifferenzierung. Zool. 
Jahrb. Abt. Ally. Physiol.. 64 : 1-13. 

HAUENSCHILD, C., 1954. Genetische und entwichlungphysiologische Untersuchugen iiher Inter- 
sexualitat und Gewebevertraglichkeit bei Hydractinia echinata Flem. H'ilhelin l\/n/.\-' 
Arch. Entwicklungsmech. ( >r<ianisiiicn. 147: 1-41. 

HUMPHREYS, T., 1963. Chemical dissolution and in Titro reconstruction of sjionge cell adhe- 
sions: Isolation and functional demonstration of the components involved. Develop. 
Biol, 8 : 27-47. 

IVKER, F. S., 1967. Localization of tissue incompatibilty of the overgrowth reaction in Hy- 
dractinia echinata. Hiol. />//.. 133 : 471-472. 

KARAKASHIAN, S., AND R. MILKMAX, 1967. Colony fusion compatibility types in Botryllns 
schlosseri.' Hil. A'////.. 133: 473. 

MILKMAN, R., 1967. ( ienetic and developmental studies on Botrvllns schlosseri. liiol. Hull., 
132: 229-243. 

MOSCONA, A., 1957. The development in vitro of chimeric aggregates of dissociated embryonic 
chick and mouse cells. Proc. Nat. Acad. Sci.. USA, 43 : 184-194. 

MULLER, W., 1964. Experimentelle Untersuchungen iiher Stockentwicklung, Polypendifferen- 
zierung und sexual Chimaren hei Hydractinia echinata. II ihclni Roti.r' Arch I:nt- 
wicklungsmech. Organismen., 155: 181-268. 

SCHIJFSMA, K., 1935. Observations on 1 1 yd rue tin in echinata ( Klein. ) and Enpai/iirus hcrn- 
/Kirdns (L). . Irch. Xccrhimt. Zool., 1 : 261-314. 

SCHIJFSMA, K., 1 ( '3 ( '. Preliminary notes on early stages in the growth of colonies of Hy- 
dractinia echinata (Flem.). Arch. Xccrlnnd. /.nol.,4: ( '3-l()2. 

So,\ XKBOKN, T. M., 1 ( '48. The determination of hereditary antigenic differences in genically 
identical Paramecium cells. I'roc. Nat. Acad. Sci. USA. 34: 413-418. 

STKI. \BEKG, M. S., 1962a. On the mechanism of tissue reconstruction by dissociated cells. I. 
Population kinetics, differential adhesiveness, and the absence of directed migration. 
Proc. Nat. Acad. Sci.. USA, 48: 1577-1582. 

STEINBERG, M. S., 1962b. Mechanism of tissue reconstruction by dissociated cells. II. Time- 
course of events. Science. 137 : 762-763. 

STEINBERG, M, S., 1963. Reconstruction of tissues by dissociated cells. Science, 141 : 401-408. 

TKISSIKK, L., AND G. TEISSIER, 1927. Les principales etapes de developpement d'l fydractiniti 
echinata ( Flem.). Bull. Soc. Zool. France. 52 : 537-547. 

TEISSIER, G., 1929. L'Origine multiple de certaines colonies d'Hydractinia echinata (Mem.), 
et ses consequences possibles. Bull. Soc. Zool. France, 54 : 645-647. 

TOTH, S. E., 1966. Polyp hypertrophy as an expression of aging in the colonial marine 
hydroid Hydractinia echinata. J. Gerontol.. 21 : 221-229. 

TOTH, S. E., 1967. Tissue compatibility in regenerating explant from the colonial marine 
hydroid Hydractinia echinata (Flem.). /. Cell I'hysiol., 69: 125-131. 

Ton \KS, P., AND J. HOLTFRETER, 1955. Directed movements and selective adhesion of em- 
bryonic amphibian cells. /. E.\-p. Zool., 128: 53-120. 



Reference: Riol. Hull.. 143: 175-183. (August, 1972) 



SPATIAL DISTRIBUTION OF NUCULE PROXLMA SAY (PROTO- 
BRANCHIA ) : AN EXPERIMENTAL APPROACH 

JEFFREY LEVINTON 

Department of Earth and Space Sciences, State University of \ci^ ) ork. 
Stony Hrook. Ncu' York 11790 

The spatial patterns of populations are influenced by many of the important 
physical and biological parameters that control the abundance and behavior of 
individuals of the population. For instance, strong environmental heterogeneity 
tends to cause a clumped distribution. This is especially true of rare species, since 
their preferred habitat is likely to occur patchily in the overall biome (Hairston, 
1959). Strong negative interactions between individuals due to direct interference 
or territoriality tend to cause a uniform distribution (Connell. 1963; Holme, 1950; 
Johnson, 1959; and others). Random distributions may result from random 
settling of larvae from the water column ( Connell, 1955 ), an abundance of resources 
(Hairston. 1959), random movements of individuals of the population, or 
a random distribution of resources. Because of the patchiness of most natural 
environments, most populations are aggregated in their spatial pattern. The 
classic study of Holme ( 1950) showed territoriality in a deposit-feeding Tellinacean 
bivalve. 

Niicnla [>ro.vinni Say (Protobranchia), an infaunal deposit-feeding bivalve was 
studied in both the laboratory and the field. This species was studied in the 
laboratory by the use of a technique employing x-radiography. By x-raying trays 
of sediment containing individuals of this species all individuals can be located 
exactly. Also, substratum heterogeneity, water characteristics, size of individuals 
and population density can be manipulated. All of these factors make laboratory 
studies of spatial patterns, where possible, highly desirable as complementary data 
to field studies. 

METHODS AND MATERIALS 

Approximately 50CO individuals of Nitcula pro.viina were collected with their 
native substratum from a depth of 20 m in Long Island Sound, off Milford, 
Connecticut. Individuals of the populations collected were of a size and morphology 
confined to very high silt-clay sediments in Long Island Sound and Buzzards Bay. 
This species is probably different from those forms living in sandy sediments of 
Buzzards Bay, Massachusetts (Hampson, verbal communication). Individuals 
were acclimated for two weeks or more in Instant Ocean Aquaria at 15 C, using 
Long Island Sound sea water (salinity 29/<>). This experimental substratum 
had a silt-clay content of over 90%, typical of natural Xncu/a substrata in Long- 
Island Sound and Buzzards Bay, Massachusetts. 

For each experiment, square trays 7.5, 10 or 15 cm on a side (0.5 cm deep) 
were filled with homogenized sediment. Niicnla was then placed on the surface 

175 



176 JEFFREY LEVINTON 

of the mud. Initial movements and subsequent vertical and lateral burrowing 
quickly eliminated the initial spatial pattern (which was usually aggregated). 
The range of densities approximated 5 X 10 : per square meter: the mean density 
for Station R, Buzzards Bay. Massachusetts (Sanders, 1960). 

Trays were kept in 25 gallon recirculating Instant Ocean Culture Systems. 
Water movements were minimized with plexiglas partitions in the aquarium. After 
an elapsed time of 10-89 days a tray was removed and placed on a piece of Kodak 
Industrial x-ray film, and x-rayed vertically (in air) with a medical x-ray unit. 
Because the bivalve shell is relatively opaque to x-rays, the positions of individual 



1 3 




cm '.._, , , J 

FIGURE 1. X-radiograph of a dispersion experiment. 

are clearly recorded on lilm ( Fig. 1). This technique was developed by D. C. 
Rhoads (see Rhoads and Young, 1970; Stanley, 1970) for the in situ study of 
infannal species. 

The x-radiograph of each tray, regardless of size, was divided into 64 quadrats 
and the number of animals per quadrat were counted. The fit of the observed pat- 
terns with a Poisson distribution was tested by using the Chi-Square goodness- 
of-fit test. 

In addition, two distance measures were used to test for departures from 
random patterns. One was the distance from an animal to its nearest neighbor 
(Clark and Evans, 1954). The second was the distance from a randomlv located 



BIVALVE SPATIAL DISTRIBUTION 177 

point to the nearest animal ( Morisita, 1954; Cottam and Curtis, 1956). If the 
population is randomly arranged, then the mean distance of this parameter should 
be the same as for the distance from an individual to its nearest neighbor (Morisita, 
1954). A test of significance suggested by Thompson (1956) was employed in 
both cases. 

For a field comparison, Nucitla proximo.- was collected at "Station R," Buzzards 
Bay, Massachusetts (Sanders, 1960). A multiple-tube sampler, similar to that 
of Buzas (1968), was constructed by cementing plexiglass cylinders of circular 
cross-section (diameter = 4.7 cm) so that the sampler consisted of 36 contiguous 
tubes, arranged in a square pattern (Fig. 2). To collect the sample, the sampler 
was inserted in the bottom. It was then capped with a cover made of foam rubber 
backed by plexiglass, and then pulled from the bottom. The suction created by the 
cap prevented the sediment in the tubes from dislodging. 

The samples were collected on August 9 and 19, 1968. The first set was 
sieved through a 0.71 mm sieve, while the second was sieved through a set of 
four sieves (2.00, 1.41, 0.70, and 0.42 mm) in order to investigate the differences 
in spatial distribution between juveniles and adults (Buzas, 1968; Jackson, 1968). 
In both samples some of the individual core samples were lost due to the difficulty 
of getting 36 cores at once. 

RESULTS 

The spatial patterns and experimental condition of the 13 analyses performed 
are shown in Table I. These experiments were done under different elapsed times. 
All x-rays but one show that the spatial pattern of Niiciila proximo, is random, 
with no territorially or gregariousness detected. The exception shows an aggre- 
gated pattern. Upon inspection of the x-rays, it was obvious that there was sig- 
nificant movement of animals to one side or corner of the tray, rather than aggrega- 
tion into small clusters. The aggregation is on a very large scale ; equal in magni- 
tude to the size of the tray. Therefore, the aggregated pattern is probably due to 
failure to homogenize the experimental environment, rather than gregariousness in 
Nucula proximo,. 

One experiment was tested for the effect of change in quadrat area. The tray 
was divided into 32, 64, 128, and 256 quadrats. In all cases, when compared to a 
Poisson distribution, the distribution of abundances did not differ significantly from 
randon (Table II). 

Using a method proposed by Clark and Evans (1954), the distance of each 
individual to its nearest neighbor was measured by determining coordinates for 
tlu- animals' locations, and measuring the distance from a point to its nearest 
neighbor. This was done by use of a computer program (on file) which selected 
the minimum distance of a given point to all other points in the experiment. In 
experiment number 10, the mean distance was found to be significantly greater 
(P < 0.05) than would be expected in a random distribution (Thompson, 1956). 
In other words this test indicated uniformity in the populations' spatial pattern. 
However, the pattern of experiment number 11 (same tray, 47 days later) did 
not differ significantly from random (P < 0.05). 

In experiment number 10, and in cases where nearest neighbor analyses of 
randomly constructed patterns proved to be uniform, the frequency distribution of 



178 



JEFFREY LEVINTON 



TABLE I 

Conditions of the experiments and resultant spatial patterns determined by 
comparison of 64 quadrat results with Poisson distribution 



Expt. # 


Elapsed 
time 
(days) 


# of 
animals 


Tray 

side 
(cm) 


Area 
(m) 


#/m 2 


Mean 
temp. C 


Com- 
puted 

X 2 


(P =0.95) 


Spatial pattern 
R = random 
A =aggregated 
U = uniform 


1 


79 


87 


15.0 


0.0225 


3.9 X 10 3 


10.0 


0.0385 


7.815 


R 


2 


32 


90 


15.0 


0.0225 


3.9 X 10' 


9.9 


0.5485 


7.815 


R 


3 


10 


92 


15.0 


0.0225 


4.0 X 10" 


6.2 


1.2993 


7.815 


R 


4 


29 


96 


15.0 


0.0225 


4.3 X 10 :i 


19.3 


0.2499 


7.815 


R 


5 


29 


97 


15.0 


0.0225 


4.3 X 10 3 


19.3 


3.5965 


7.815 


R 


6 


89 


92 


10.0 


0.0100 


9.2 X 10 3 


9.5 


0.8137 


7.815 


R 


7 


10 


225 


15.0 


0.0225 


1.0 X 10 4 


6.2 


3.2413 


11.143 


R 


8 


35 


117 


10.0 


0.0100 


1.2 X 10 4 


19.8 


1.6825 


11.143 


R 


9 


35 


124 


10.0 


0.0100 


1.2 X 10 4 


19.8 


5.4929 


7.815 


R 


10 


32 


298 


15.0 


0.0225 


1.3 X 10 4 


9.9 


5.2118 


12.592 


R 


11 


79 


298 


15.0 


0.0225 


1.3 X 10 4 


10.0 


3.6320 


12.592 


R 


12 


89 


222 


10.0 


0.0100 


2.2 X 10 4 


9.5 


1.9495 


11.071 


R 


13 


10 


132 


7.5 


0.0563 


4.1 X 10 4 


6.2 


12.5868 


11.143 


A 



nearest neighbor distances was right-skewed. However, in the patterns not differ- 
ing significantly from the Clark and Evans null hypothesis of randomness, this 
frequency distribution was closer to normal. The statistical test proposed by Clark 
and Evans (1954) requires such a normal frequency distribution. 

A similar test involves locating points randomly within the area in question, and 
measuring the distance from each point to the nearest animal (Morisita, 1954; 
Cottam and Curtis, 1956). Using this test on experiment 11, the mean distance 
was not fourra to differ from that mean distance expected in a random distribution 
(P<0.05). 

The point-centered quarter method was also employed on this experiment. 
This consists of dividing an area around randomly selected points into quadrats. 
The distance from the point to the closest animal in each quadrat is measured. 
This method showed the spatial pattern of this experiment to not differ significantly 
from random (P < 0.05). 

Spatial patterns, particularly uniform ones, are strongly affected by the variance 
in body size of the population. Pielou (1960) showed that organisms that are 
"territorial" will yield an aggregated distribution if the range in size is so great 

TABLE 1 1 

Effect of differing quadrat areas on spatial distribution in experiment #llf 
computed x 2 is based on comparison with the Poisson distribution 



# of Quadrats 


Computed 

X 2 


x 2 , P = 0.95 


Spatial pattern 


32 


0.1271 


7.815 


R 


64 


3.6320 


12.592 


R 


128 


1.6107 


11.071 


R 


256 


3.0392 


9.488 


R 



BIVALVE SPATIAL DISTRIBUTION 



179 



as to produce a corresponding range in distance to nearest neighbors. This factor 
is inferred to operate if there is a correlation between nearest-neighbor distances 
and the sum of their sizes (Connell, 1963). Two experiments were tested for this 
effect. In both analyses (analysis l:r- -0.154, d.f. == 33, F 0.798 ; analysis 
2 : r -- 0.054, d.f. == 33, F -- 0.095) no significant correlation was found. 

In conclusion, it is judged that Nitcula pro.riiua maintains a random distribu- 
tion in a homogeneous experimental environment. 

The two field samples from station R show that the spatial pattern of Nucnla 
proximo, in its natural habitat is also random. One sample was sieved core by 
core into 4 size fractions (2.00 mm, 1.4 mm, 0.77 mm, and 0.42 mm) and these 




FIGURE 2. Multiple-coring device used in collection of Station "R" 
samples. Diameter of each core is 4.7 cm. 

groups were evaluated individually. The spatial pattern for all size fractions com- 
bined was evaluated for both sample sets. In both sample sets only 29 cores were 
recovered successfully. The results of the analyses are shown in Table III. In 
all cases but one the spatial pattern is random. The one exception, size fraction 
c, is aggregated. There are slight tendencies in all of the field samples towards 
aggregation ; being probably an indication of the heterogeneity of the environment. 
It must be remembered that the total area sampled by each core set is equal to 
about twice the area of the largest experimental tray. Thus the chances for en- 
vironmental heterogeneity are greatly increased, especially since the environmental 
area is by no means approximately homogeneous; as in the experimental trays. 
These results clearly show the need for contrasting lab and field studies of spatial 



180 



JEFFREY LEVINTON 



patterns. With laboratory investigation, one can learn those interactions which are 
due solely to positive or negative interactions between individuals ; and not to 
environmental heterogeneity. 

DISCUSSION 

Nucula pro.riina is a deposit-feeding bivalve, feeding by means of a tentacle-like 
palp proboscide which conveys detritus to the mouth by means of a ciliated groove. 
It feeds within the substratum, rather than grazing on the surface for particles de- 
livered by the overlying water. The abundance of Nucula proximo, in Buzzards 
Bay and Long Island Sound correlates with parameters related to the availability 
of food (Sanders, 1956, 1958). Therefore, populations of Nucula are probably 
food limited, and at the environment's carrying capacity. This food limitation 
might produce the expectation that N. pro.riina would be territorial (i.e. maintain 
a uniform dispersion pattern) in order to ensure a predictably available food 
source. The following arguments, however, indicate that there should be no selec- 
tion for territoriality in fairly dense populations of this species. 

TABLE III 

Analysis of polycorer data, comparing core abundance frequencies with thos^ expected 

in a Poisson distribution; using the Chi Square goodness-of-fit test. Rl taken 

August 19, 1968; R2 taken August 8, 1968. A, B, C, and D refer to 

2.0, 1.4, 0.7, and 0.4 mm fractions, respectively. Total 

populations are "/" samples 



Samples 


R1A 


RIB 


R1C 


RID 


RH 


R2t 


Calculated \- 


2.37 


1.34 


6.27 


3.61 


2.64 


0.99 


X 2 0.95 


3.84 


5.99 


5.99 


5.99 


5.99 


3.84 


Spatial pattern 


R 


R 


A 


R 


R 


R 



( 1 ) There is no known available means of territorial defense for Nucula to 
employ. In addition there is no readily available mechanism to allow this species 
to sense and interact with other individuals of the population. Unlike species of 
Tellina (Bivalvia: Tellinacea), which have long, mobile siphons, Nucula com- 
municates with the outside medium only with its relatively short palp proboscides 
and has no siphons. In addition, Nucula maintains no permanent burrow. 

( 1 } Furthermore, consider a fairly dense population of deposit-feeders who 
move about constantly. When an individual leaves his former area of foraging, 
he has two choices He can either return to his own area, or move into the area 
of another individual. If the population is fairly dense, it is probable that the 
area of the second individual has been just as completely (or incompletely) ex- 
ploited as that of the first. Therefore, there is no relative advantage or disad- 
vantage to turning about and feeding upon a home territory or continuing into 
another individual's feeding area. This is especially true as Nucula can only feed 
on a relatively small area at a time, and has to keep burrowing around. This is 
in contrast to deposit-feeding Tellinacean bivalves, who in stationary life position 
can feed on a relatively large area with their long, mobile siphons. In this case 
it is energetically more economical to maintain a territory and stay in one place 



BIVALVE SPATIAL DISTRIBUTION 181 

(since the feeding area is large) than to move about. This is especially true for 
those forms whose food source is delivered by currents to the bottom. These above 
arguments probably explain why Tel Una tennis and T. ayilis seem to be territorial 
and maintain uniform distributions (Holme, 1950; William Gilbert, verbal com- 
munication ) ; whereas Nucula proximo' s spatial pattern is apparently random. 

(3) A third argument leading to the expectation of randomness in Nucula 
spatial patterns involves the stability and water content of the sediment. The 
reworking activities and fecal pellet formation of Nucula proximo, increases the 
water content of the sediment and produces an unstable, fluid medium (see Rhoads 
and Young, 1970; Levinton, 1971 ). In the presence of weak bottom currents the 
fluid substrate is easily suspended, making the stability of Niicula's living position 
very unpredictable. Under these conditions, it is unlikely that selection for be- 
havior relating to territoriality would be at all important. 

There is no difference in the spatial patterns of small vs. large individuals in 
the field samples. Others have found that juveniles are often aggregated, whereas 
adults are random or uniform. Jackson (1968) and Buzas (1968) ascribed this 
to parental release of offspring, leading to parent-offspring aggregations. Green 
and Hobson (1968) claim that this initial aggregation, in the case of Gemma 
(/ciuina (Bivalvia, studied by Jackson, 1968) would be soon eliminated by tidal 
currents, or would be altered by substrate heterogeneity. Gilbert (1968) found 
these same dispersion phenomena in natural populations of the Dward Tellin Clam, 
Tcllina in/ilis. He demonstrated that the settling velocity, and subsequent aggre- 
gation, of juveniles was identical to the settling velocity of sand 0.2 mm in diameter. 
Sediment of this mean diameter was the substratum of maximum abundance of 
Tell'ma juveniles. Studies of juvenile vs. adult spatial patterns are clearly impor- 
tant sources of data relating to the change of selective forces with increasing age. 

Connell (1963) examined and compared the spatial patterns of several marine 
benthic invertebrate populations, of widely differing ecological types. He noted 
that Uca [>it</ilator, an epifaunal deposit-feeding fiddler crab, and Erichthonius 
braziliensis , an epifaunal grazing amphipod, both maintain uniform spatial patterns. 
Individuals of both species live in permanent dwellings and the need for the defense 
of territory is obvious. As Connell (1963) also argues, the establishment of 
territories allows all of the energy of the individual to be channelled into growth 
and reproduction. Nncnla proximo.-, and other mobile infaunal deposit-feeders, 
do not fit into this category since they do not maintain permanent burrows. In 
addition, their mode of feeding is different from Tcllina. which Connell also lists 
as having a uniform pattern. 

In infaunal suspension feeding populations environmental heterogeneity seems 
to be very important in bringing about clumped patterns (Connell, 1963). The 
factors which determine the abundance of suspension-feeders are those which often 
are not systematically distributed in space, or predictable in time. This tends to 
lead to aggregated distributions. In contrast, deposit-feeders' spatial patterns are 
strongly determined by their feeding and burrowing behavior. Deposit-feeders 
tend to condition their substratum, and control its structure (Rhoads and Young, 
1970). 

The above makes it clear that it is difficult to make comprehensive statements 
about the spatial distribution of organisms. It is apparent that spatial patterns are 



182 JEFFREY LEVINTON 

controlled by the interaction of environmental heterogeneity, trophic group, mode 
of reproduction and mobility. However, spatial patterns are invaluable in the 
investigation of specific problems relating to behavior, environmental heterogeneity 
and the environmental components of selection. 



I am grateful to I). C. Rhoads for provision of equipment, much helpful dis- 
cussion and continued encouragement. Conversations with W. H. Gilbert were 
important in the development of this study. Roger Green read the manuscript. 
I thank J. B. Howard and D. K. Young for collecting samples at Station "R." 
Finally, I thank H. L. Sanders (Woods Hole Oceanogr. Inst.). whose provision 
of lab space at Woods Hole made the initiation of this study possible. This study 
was supported by a Penrose Bequest (Geol. Soc. America) and by a Sigma Xi 
grant-in-aid. 

SUMMARY 

The spatial patterns of populations of Nncula proximo, were studied in the 
laboratory using x-radiography, and in the field using a multiple-coloring device. 

Trays with sediment and varying population densities of N. proximo, were 
x-rayed under different temperature and elapsed time conditions. Almost all 
experiments were shown to exhibit random patterns. The two exceptions were 
aggregated distributions which were probably due to inhomogeneities in the experi- 
mental environment. 

The field samples showed Nncula proximo- to be randomly distributed, with a 
tendency towards aggregation in some cases. Juveniles were distributed essen- 
tially the same as the adults. 

It is argued that the lack of defense mechanisms, the instability of the substrate, 
the small "reach" of the feeding organ, and the lack of advantage of territoriality to 
a mobile deposit-feeder all contribute to the observed random patterns of Nncula 
proximo,. 

It is concluded that x-ray studies of infaunal invertebrates done in concert with 
field studies is an excellent means of distinguishing the factor of environmental 
heterogeneity from negative or positive interactions between individuals caused 
by territoriality or gregariousness. 

LITERATURE CITED 

BLACKRITH, R. E., 1958. Nearest neighbor distance measurements for the estimation of ani- 
mal populations. Ecology, 39: 147-150. 

BUZAS, M. A., 1968. On the spatial distribution of foraminifera. Coutr. Citsliinan Found. 
Foram. Res., 19 (pt. 1, no. 342) : 1-11. 

CLARK, P. J., AND F. C. EVA\\S, 1954. Distance to nearest neighbor as a measure of spatial 
relationships in populations. Ecology, 35 : 445-453. 

CON NELL, J. H., 1955. Spatial distribution of two species of clams, Mya arciuiria L. and Pctri- 
cola pholadifonnis Lamark, in an intertidal area. Rep. Fish. hi-c. Mass., 8: 15-25. 

COISTNELL, J. H., 1963. Territorial behavior and dispersion in some marine invertebrates. Res. 
Pop.Ecol.,S: 87-101. 

COTTAM, GRANT, AND J. T. CURTIS, 1956. The use of distance measures in phytosociologic I 
sampling. Ecology, 37: 451-460. 



BIVALVE SPATIAL DISTRIBUTION 183 

COTTAM, GRANT, J. T. CURTIS AND B. W. HALE, 1953. Some sampling characteristics of a 
population of randomly dispersed individuals. Ecology, 34: 741-757. 

GILBERT, W. H., 1968. Distribution and dispersion patterns of the dwarf tellin clam, Tcllina 
agilis. Biol. Bull, 135: 419-420. 

GREEN, R. H., AND K. D. HOBSON, 1968. Factors determining spatial and size frequency dis- 
tributions of Gemma gemma. Science. 162: 1509-1510. 

HAIRSTON, N. G., 1959. Species abundance and community organization. Ecology, 40: 404- 
416. 

HOLME, N. A., 1950. Population dispersion in Tcllina tennis da Costa. /. Mar. Biol. Ass. 
U. K., 29 : 267-280. 

JACKSON, J. B., 1968. Bivalves : spatial and size frequency distributions of two intertidal spe- 
cies. Science. 161: 479-480. 

LEVINTON, J. S., 1971. The ecology of shallow water deposit-feeding communities. Ph.D. 
dissertation, Yale University, 284 pp. 

MORISITA, MASAAKI, 1954. Estimation of population density by spacing method. Mem. Fac. 
Sci. Kyushu Univ.. Series E, 1: 187-197. 

OWEN, D. B., 1962. Handbook of Statistical Tables. Addison- Wesley, Reading, 580 pp. 

PIELOU, E. C., 1960. A single mechanism to account for regular, random, and aggregated 
populations. /. Ecol., 48: 575-584. 

PIELOU, E. C., 1969. An Introduction to Mathematical Ecology. Wiley-Interscience, New 
York, 286 pp. 

RHOADS, D. C., AND D. K. YOUNG, 1970. The influence of deposit-feeding benthos on bottom 
sediment stability and community trophic structure. /. Mar. Res., 28: 150-178. 

SANDERS, H. L., 1956. Oceanography of Long Island Sound, 1952-1954. X. Biology of marine 
bottom communities. Bull. Bingham Oceanogr. Coll., 15: 345-414. 

SANDERS, H. L., 1960. Benthic studies in Buzzards Bay. III. The structure of the soft bottom 
community. Limnol. Oceanogr., 5: 138-153. 

STANLEY, S. M., 1970. Relation of shell form to life habits of the Bivalvia (Mollusca). Geol. 
Soc. America, Mem., 125: 1-296. 

THOMPSON, H. R., 1956. Distribution of distance to nth neighbor in a population of randomly 
distributed individuals. Ecology , 37: 391-394. 



Reference: Biol. Bull,, 143: 184-195. (August, 1972) 



CHEMORECEPTION IN THE MIGRATORY SEA TURTLE, 

CHELONIA MY DAS 

MARION MANTON, ANDREW KARR AND DAVID W. EHRENFELD 

Departments of Biological Sciences and Psychology, Columbia University, New York, 

Neiv York 10027 and Department of Biological Sciences, Barnard College, 

Columbia University, New York, Nczv York 10027 

It has long been suspected on the basis of neuroanatomical (Papez, 1961), 
neurophysiological (Tucker, 1963; Tucker and Shibuya, 1965) and behavioral 
evidence (Boycott and Guillery, 1962), that fresh water and land turtles have a 
sense of smell. Nothing is known of chemoreception in sea turtles, although logger- 
head turtles (Caretta caretta} have been observed underwater with their nostrils 
open and the floor of the mouth moving up and down, possibly engaged in chemical 
sampling (Walker, 1959). 

The green turtle (Chelonia mydas), whose life cycle has been studied inten- 
sively, is known to migrate long distances through the open sea. Tagging studies 
have demonstrated that populations of Atlantic green turtles usually leave their 
year-round feeding grounds to mate and breed on beaches that are hundreds of 
miles away (Carr, 1967). For example, the population that nests on Ascension 
Island feeds near the coast of Brazil, a distance of 1400 miles. Their method of 
navigation is unknown. Orientation by visual cues alone seems unlikely, moreover 
these turtles have been shown to be myopic when their eyes are out of water 
(Ehrenfeld and Koch, 1967). It has been suggested recently that the detection 
of chemicals entering the South Equatorial Current from Ascension Island might 
aid in the navigation of the Brazilian migrants (Koch, Carr and Ehrenfeld, 1969). 
Carr (1972) has called attention to evidence that olfactory cues might also be 
available to migrants to a mainland nesting shore. 

In this study we used operant conditioning techniques to examine the ability 
of the green turtle to detect various chemical substances dissolved in water. In 
addition we tested a method of reversibly interrupting olfaction for a period of 
days by treating the olfactory epithelium with a 0.35 M solution of ZnSO 4 . 

MATERIALS AND METHODS 

The experimental subjects were four immature Caribbean green turtles. At 
the start of the experiment they were 6 months old and weighed 300 to 450 g. 
They lived in recirculating artificial sea \vater and were tested in fresh running 
water (green turtles are osmotically highly adaptable). The turtles had been 
hatched and reared in the laboratory from eggs obtained in Costa Rica, and were 
previously untested. They were kept on a 23-hr food deprivation schedule during 
the experiments. 

The apparatus used for training and testing is diagrammed in Figure 1. The 
experimental chamber was a tank 30 cm wide, 45 cm long and 30 cm deep con- 
taining water at a depth of 7 cm (81) which flowed continuously through the 

184 



CHEMORECEPTION IN SEA TURTLE 



185 




FIGURE 1. Diagram of the experimental tank; (a) chemical or water reservoir; (b) re- 
lease valves; (c) glass conduit housing delivery tubes; (d) turtle pressing left (signal 
production) key; (e) second reservoir; (f) automatic feeder; (g) overhead light; (h) water 
inlet; (i) key light ; ( j ) water level ; (k) one of the three water outlets. 



186 M. MANTON, A. KARR, AND D. W. EHRENFELD 

tank at a controlled temperature of 26 C and a rate of 8 1/min. The turtles were 
able to swim about freely, with their heads submerged. Two response keys 4 X 
5 cm in size and 8 cm apart were suspended under water at one end of the tank 
in the path of the water inflow. A light was mounted above water level over each 
key. An overhead light provided general tank illumination. An automatic feeder 
was positioned over the right key. The experiment was programmed by standard 
relay equipment housed in a separate room. Data were recorded automatically 
on counters. A masking noise was provided in the experimental room to eliminate 
possible cues from apparatus sounds. 

The turtles were trained in a series of steps, using techniques modified from 
Nevin (l l )/0) and described in detail elsewhere (Manton, Karr and Ehrenfeld, 
in press ). Briefly, the turtles were acclimatized to the tank until food was accepted, 
then they were required to press the right key with their heads in order to obtain 
a food reward (small cubes of meat, which were dropped automatically into the 
tank). Subsequently they were trained to press first the left key, then the right 
key before reinforcement was delivered. During this initial training a light signal 
was the cue for reinforcement availability. When this task was learned the light 
was illuminated on an intermittent schedule. Soon the turtles were observed to 
press the left key almost continuously, while watching the light over that key. 
After this light was turned on, if the turtle then responded to the right key within 
20 sec, reinforcement was delivered. After each correct response to the right 
key the light above that key was illuminated for 1 sec. This light acted as a 
secondary reinforcer, signalling the delivery of food to the tank. 

When the turtles had acquired stable behavior patterns and correct responding 
was at the 90% level, the intensity of the signal light was progressively reduced 
to zero and the chemical signal was simultaneously introduced in its place. Thus, 
in the final procedure, the turtles worked steadily at pressing the left key. When- 
ever a response at this key resulted in the underwater release of chemical, the 
turtle was given 20 sec in which to press the right key for a food reward. 

Although the use of two underwater keys complicated the training procedure, 
responses to the left (signal production) key maintained attention to the stimulus 
and insured that the turtle's head would always be near the chemical release point 
upon signal presentation. The right (reinforcement) key served as the reporting 
key when a stimulus was detected. The tendency to respond occasionally to this 
right key when no stimulus was present was reduced by the necessity of activity 
at the left key, and was further discouraged by the introduction of a 2-sec blackout 
period (mild negative reinforcement) following each false report. 

After training, each experimental session consisted of 50 trials, 25 with 0.5 ml 
of the test chemical and 25 with 0.5 ml of a water control, presented in a quasi- 
random sequence. Two separate delivery systems released the test liquids to the 
tank underwater. These systems were constructed of glass tubing ; solenoid- 
operated valves controlled the flow of the contents of the two delivery tubes. 
Both tubes released their contents into a common conduit which extended just 
below the surface of the water near the left hand response key (see Fig. 1 ). The 
test liquids were occasionally alternated between the two delivery systems. 

The first response to the left key after a minimum time of 1 min had elapsed 
from the previous trial, started a new trial. During the 20 sec after chemical 



CHEMORECEPTION IN SEA TURTLE 187 

presentation, a response to the right key was scored as a correct report and rein- 
forcement was delivered. During the 20 sec after water presentation, a response 
to the right key was scored as a false report and no reinforcement was delivered. 
The false reports were used as controls to sample the tendency to respond to the 
reinforcement key when no signal was present. 

The first test substance was 0.05 M /3-phenethylalcohol. This chemical has 
been used in olfactory threshold studies with teleost fish (Teichmann, 1959) and 
is non-toxic, non-irritating and colorless at the concentration used. The interval 
between chemical trials varied from 1 to 4 min during a session. A minimum 
interval of 1 min was chosen because tests with an indicator dye added to the tank 
water in the same concentration as the test chemical, showed a 9S c /c reduction 
from the initial concentration during the first min after dye addition. 

The other organic chemicals tested were also selected on the basis of their use 
in previous experiments on chemoreception. They were, in order of their presenta- 
tion: 0.05 M iso-pentyl acetate, 0.01 M triethylamine, 0.01 M cinnamaldehyde, 0.1 M 
L-serine and 0.1 M glycine. (Calculations of actual concentrations in the tank at 
the time of chemoreception are presented in "Discussion.") The procedure was 
identical in all cases. Data were collected from a minimum of 10 consecutive 
sessions for each test chemical. 

The experiment, as outlined, cannot differentiate chemical discrimination medi- 
ated by olfaction from that mediated by taste. Therefore the method developed 
by Alberts and Galef ( 1 ( ->71 ) for producing temporary anosmia in rats by bathing 
the olfactorv mucosa with ZnSO, solution, was modified for use with these marine 

J 

turtles. Reagent grade ZnSO 4 7H,O was used in making solutions. After test- 
ing several concentrations we selected 0.35 M ZnSO 4 for use. 

Before treatment with either ZnSO 4 or a control solution (NaCl or MgSO 4 ) 
each turtle was removed from the home tank for at least one hour to permit drying 
of the nasal cavities and month. The turtle was then placed on its carapace with 
its head tilted downwards. The mouth was held open, and the tongue was screened 
from contact with the ZnSO, during treatment. The solution was injected, using 
a recurved and blunted syringe needle, directly into the internal nares. Approxi- 
mately 0.3 cc was introduced on each side ; drops were observed to run out of the 
external nares. The area around the internal nares was aspirated to remove 
any excess ZnSO 4 and the turtle was kept in the same position for a few minutes 
to prevent solution from draining back into the mouth. The turtle was returned 
to the home tank an hour after treatment. Two turtles (Nos. 2 and 3) were 
treated with 0.35 M ZnSO 4 solution in this manner. The other pair received an 
identical intranasal injection of either 0.35 M NaCl (No. 1) or 0.35 M MgSO 4 
(No. 4) as controls. 

Turtles were run in their usual chemical discrimination test sessions on the 
same day as treatment and daily thereafter until behavior returned to the pre- 
treatment baseline. 

The treatment was then repeated with the modification that the intranasal 
injection was made through the external nares. Care was taken to ensure that the 
head tilted downwards throughout the treatment to minimize flow to the mouth; 
the mouth was opened, and the solution was injected until it welled up in the 
internal nares and in the nostril not being injected. The previous control animals 



188 



M. MANTON, A. KARR, AND D. W. EHRENFELD 



TABLE I 
Mean responses ( c : c ) to 4 chemicals and to water, averaged over 15 sessions 





^-phenethylalcohol 


iso-pentyl acetate 


triethylamine 


cinnamaldehyde 


Turtle 

Mr* 


Mean 


Mean 


Mean 


Mean 


Mean 


Mean 


Mean 


Mean 




correct 


false 


correct 


false 


correct 


false 


correct 


false 




detection 


reports 


detection 


reports 


detection 


reports 


detection 


reports 




(%) 


(%) 




(%) 


(%) 


(%) 


(%) 


(%) 


1 


75.7 


32.3 


86.1 


45.3 


87.5 


42.1 


81.6 


28.5 


2 


93.1 


42.1 


96.3 


65.6 


94.9 


57.3 


94.4 


47.2 


3 


85.9 


23.2 


92.3 


52.5 


95.7 


49.6 


92.5 


45.9 


4 


77.3 


14.4 


85.3 


39.2 


93.1 


44.0 


92.0 


37.1 


Average 


83.0 


28.0 


90.0 


50.7 


92.8 


48.3 


90.1 


39.7 



were injected with ZnSO 4 solution (Nos. 1 and 4) while the other pair, which 
had recovered full olfactory function, received identical injections of NaCl (No. 2) 
andMgSO 4 (No. 3). 

RESULTS 

Once trained, the green turtles maintained a steady base rate of response to 
the left (signal production) key with an average of 10 responses/min. Neverthe- 
less, the tendency to respond occasionally to the food key when no signal was 
present was never completely eliminated. 

Approximately 30 sessions/turtle were required to effect the transfer of the 
learned operant behavior from the progressively reduced light signal to the first 
chemical signal employed (0.05 M /? -phenethylalcohol). By the time the light was 
dimmed to almost zero intensity the turtles had ceased to look at it, as was their 
previous habit. The results from 15 consecutive sessions with phenethylalcohol 
after signal pairing was discontinued are graphed in Figure 2A. The open circles 
of each graph show the c /c correct reports after chemical release and the open 
squares show the % false reports after water release. The turtles all responded 
to the right (food) key in the presence of the phenethylalcohol solution with a 
consistently higher probability than to the water control. The mean performance 
for the phenethylalcohol trials for all 4 turtles, averaged over the 15 sessions (a 
total of 1500 trials), was 83% correct detection. The mean performance for the 
same number of water control trials during the same 15 sessions was 28 % false 
reports. The individual means are included in Table I. 

Similar results were obtained when the test chemicals were 0.05 M isopentyl 
acetate, 0.01 M triethylamine and 0.01 M cinnamaldehyde. In these cases, the turtles 
readily generalized the experimental function of "chemical" and retraining was not 
necessary when a new test substance was introduced. The per cent correct detec- 
tion of the test chemical and the per cent false reports to the water tended to 
vary together. This covariance indicates that although absolute detection varied, 
relative detection was fairly stable. This was largely a function of a turtle's general 
activity level on a particular day. 

Table I summarizes the data for the first 4 chemicals tested. Each entry in 
Table I reports the per cent response by an individual turtle to 375 chemical or 
water trials during 15 consecutive sessions. 



CHEMORECEPTION IN SEA TURTLE 



189 



TABLE II 
Mean responses (' , ) to 2 amino acids and to water, averaged over 10 sessions 



Turtle N"o. 


L-Serine 


Glycine 


Mean 
correct 
detection 
(%) 


Mean 
false 
reports 
(%) 


Mean 
correct 
detection 
(%) 


Mean 
false 
reports 
(%) 


1 


65.7 


71.6 


80.8 


80.0 


2 


84.4 


84.8 


85.6 


86.0 


3 


85.2 


82.4 


86.0 


84.6 


4 


74.8 


75.2 


79.2 


68.0 


Average 


77.5 


78.5 


82.9 


79.7 



Direct observation of the turtles during their daily sessions revealed a distinctive 
change in their otherwise leisurely behavior, upon release of the chemical. Flipper 
movements markedly increased and approaches to the reinforcement key were often 
quite violent. At times, after chemical release, the turtles became too excited to 
push the key within the 20 sec limit allotted for correct detection. This frenzied 
behavior was not observed during water trials. The entire behavioral sequence 
of pressing on the left key, swimming over to the right key and pressing it, invari- 
ably occurred with the head completely under water. During chemical release the 
turtles directed their nostrils downward and appeared to be pumping water through 
the nasal cavities by means of throat movements. Breathing pauses, during which 
the nostrils were above water, were infrequent and were only made during well 
defined breaks in responding. 

As a session progressed we were able to detect a slight odor of the test 
chemical above the experimental tank. However, it appears virtually impossible 
that the turtles w r ere using this odor as a cue since the correlation between airborne 
odors, reinforcement availability and the emergence of the nostrils above water 
was necessarily random. 

The results of the tests of the two amino acids, L-serine and glycine, are given 
in Table II, and the L-serine results are graphed in Figure 2B. Trials were stopped 
after 10 consecutive sessions because the learned response pattern was disintegrat- 
ing in the absence of any stimulus that the turtles could discriminate. It was 
necessary to retrain the turtles to baseline performance after each amino acid was 
tested. Phenethylalcohol, earlier shown to be detected, was used for this purpose. 

ZnSO 4 solution temporarily interrupted chemical discrimination for periods 
lasting from 1 to 5 days. Recovery of function occurred gradually over a period 
of several days. Chemoreception before and after ZnSO 4 treatment can be com- 
pared for all 4 turtles in Figure 3 A. Turtles 1 and 4 received ZnSO 4 through 
the external nares and the resulting anosmia was brief. The intranasal injections 
of saline (turtles 1 and 2) and MgSO 4 solution (turtles 3 and 4) had virtually 
no effect on the performance of the chemical discrimination, as shown in Figure 3B. 
None of these localized chemical treatments caused any other observed behavioral 
changes. Turtles treated with ZnSO 4 swam and fed normally immediately after 
treatment. The discriminative test chemical was 0.01 M cinnamaldehyde. 



190 



M. MANTON, A. KARR, AND D. W. EHRENFELD 



100 
75 
50 
25 


100 

75 
50 



o 25 

Q_ 




100 
75 
50 
25 


100 

75 
50 
25 



100 
75 



c PHENETHYLALCOHOL 
u WATER 




TURTLE 1 



TURTLE 3 



TURTLE 4 



TURTLE 1 









1 3 5 7 9 11 13 15 
A SESSION 



25 


100 

75 
50 
25 


100 

75 
50 
25 


100 

75 

50 

25 





o L SERINE 
WATER 




TURTLE 2 




TURTLE 3 




TURTLE 4 



1 2 3 4 5 6 7 8 9 10 
B SESSION 



Fu;rKK 2. The per cent correct and false reports for 4 green turtles during presentation 
of phenethylalcohol (A) and l.-serine (B). The open circles show the per cent correct reports 
after chemical release. The open auuares show the per cent false reports after water release. 



100 

75 
50 
25 


100 

75 
50 
25 

- 

1= 100 



o 

Q_ 



UJ 

CL 



75 
50 
25 


100 

75 
50 
25 



CHEMORECEPTION IN SEA TURTLE 

lOOr 



191 




CINNAMALDEHYDE 
WATER 



ZNS0 4 



1 3 



TURTLE 2 



ZNS0 4 



1357 



ZNS0 4 



1 3 



13579 
A SESSION 



11 13 




11 13 




11 13 




11 13 



75 
50 
25 


100 

75 
50 
25 



o CINNAMALDEHYDE 
a WATER 

TURTLE 1 



NACL 



1 2 3 4 5 6 7 8 9 10 



TURTLE 2 



NACL 



1 2 3 4 5 6 7 8 9 10 



100 
75 
50 

25 




TURTLE 3 



MGS04 



1 2 3 4 5 6 7 8 9 10 



100 



75 



50 



25 




TURTLE 4 




1 2 3 4 5 6 7 8 9 10 
B SESSION 



FiGi'Rii 3. The per cent correct (open circles) and false (open squares) reports for 
4 green turtles before and after intranasal treatment (arrow) \\itli ZnSO* (A) or either 
NaCl or MgSO 4 (B). See text for further details. 



M. MANTON, A. KARR, AND D. W. EHRENFELD 

DISCUSSION 

Our results indicate that green turtles can detect a chemical dissolved in water. 
The inability of the turtles to perform the discrimination following treatment with 
ZnSO 4 solution suggests that this chemoreception is mediated by olfaction. Thus 
these turtles are able to smell underwater, an unusual ability for an air-breathing 
vertebrate (Evans and Bastian, 1969). [For purposes of this discussion we assume 
that "olfaction" is mediated by the entire sensory epithelium of the nasal cavity, 
which includes some tissue possibly homologous with Jacobson's organ of other 
reptiles, and which is innervated by the vomeronasal nerve (Parsons, 1967).] 

There was no evidence that taste played a role in the performance of the 
chemical discriminations, although further study is indicated. In histological 
examinations of the epithelia of the palate and tongue of Chelonia, we have so far 
been unable to identify taste receptors, whereas anatomical structures associated 
with olfaction are present and well developed. In other turtles (the land tortoise, 
Gophcrus), taste receptors have been found to be small and localized to the tip of 
the tongue (personal communication, P. P. C. Graziadei, Department of Biological 
Sciences, Florida State University). 

Sensory adaptation has not proven to be a problem in our procedure. Although 
the test chemical was not completely cleared between all trials, a sudden increase 
in chemical concentration was satisfactory as a discriminative stimulus. If some 
sensory adaptation did occur between trials, despite steadily decreasing concen- 
tration, the addition of the test chemical solution at the next trial provided a suf- 
ficient change in the stimulus to act as a new signal. This was clearly confirmed 
both by the test scores, themselves, and by the directly observed behavioral changes 
which occurred after presentation of chemical stimuli. 

The sound of running water served to mask the noise of the solenoid-operated 
valves, and the control and chemical solutions were occasionally switched in any 
case so that no valve sound could serve as a reliable cue to chemical presentation. 
Relays, counters and tape programming apparatus were all located in an adjacent 
room behind a closed door, and their various sounds (similar for chemical and 
water trials ) were inaudible to us in the room with the test chamber. \Ye have 
dismissed the possibility that inadvertant apparatus sounds could function in the 
discrimination. 

Differences among the average scores for the first 4 chemicals tested, and for 
their controls (Table I), probably represent differences in the turtles' level of 
training and experience and in their strategy of response rather than differences 
in perception of the various odors. In each of these test series the fact that there 
is a large and consistent difference between correct detection and false reports is 
more significant than the exact amount of that difference. 

A fairly high rate of inappropriate responding to the reinforcement key in the 
absence of a stimulus is common in operant situations where there is little or no 
effective punishment for making false responses (Azrin and Holz. 1966). The 
2-sec blackout did not constitute strong negative reinforcement. (During training, 
when the light was the discriminative stimulus, the percentages of both false and 
correct reports were comparable to those recorded in the chemical trials.) 

The transfer from one test chemical to another presented no difficulties and 



CHEMORECEPTION IN SEA TURTLE 193 

gave clear evidence of stimulus generalization. A single session was usually suf- 
ficient to establish correct responding to the new odor. On the other hand, when 
the stimulus presented could not be detected, behavior was disrupted. The behavior 
during the amino acid test sessions was characterized by alternation between the 
keys, frequent pauses between bouts of responding and occasional defecation in 
the experimental tank. (Salmon, unlike Clicloniu. are reported to be able to de- 
tect L-serine in extremely low concentrations ( Idler, Fagerlund and Mayoh, 
1956).) 

It is possible to make an estimate of the sensory acuity demonstrated in this 
experiment. From the relative positions of the turtle (at the left key) and the 
chemical release point we can assume that the delivered chemical is diluted, at the 
time of sampling, in a volume of water equivalent to -J of the tank volume (1000 
ml). Detection therefore occurs at approximate concentrations of from 5 X 10~ 6 M 
to 5 X 10~ 5 M depending on the solution used. Dye tests confirm the assumption 
underlying this calculation. (The undetected amino acids were presented at an 
approximate concentration of 10~ 4 M.) 

The mechanism of the ZnSO 4 -induced anosmia is as yet unknown. The data 
from the saline and MgSO 4 -treated controls appear to rule out osmotic shock or 
trauma following treatment. The role of Zn ++ seems crucial. The present method 
of ZnSO 4 application produces considerable variation in the period of anosmia. 
One turtle showed definite signs of the return of olfaction after 24 hr, while the 
longest period of complete anosmia was 5 days. Factors such as the mode of 
administration and the degree of dryness of the nasal passages probably influenced 
the effectiveness of the treatment. 

Since the animals used in this study are difficult to obtain, Zn ++ -induced 
peripheral anosmia has certain advantages over olfactory bulb ablation or olfactory 
nerve sectioning. The Zn l + effect is both reversible and relatively non-traumatic. 
Furthermore, the present technique provides the opportunity for field studies of 
the role of olfaction in the orientation of green turtles, without causing permanent 
loss of functional individuals from an endangered population. The possible role 
of olfaction in both open sea navigation and in site selection at the nesting beach 
can be experimentally studied with this approach. In general, the use of Zn ++ -in- 
duced anosmia offers promise of opening new areas of investigation of the inter- 
action between olfaction and behavior among a wide range of vertebrates ; and it 
may provide an additional tool for the study of the mechanism of olfaction itself. 

Our experimental procedure is a sensitive behavioral assay for underwater 
chemoreception in aquatic vertebrates. It further demonstrates the utility of operant 
conditioning methods in the study of reptilian sensory physiology. Our findings 
show that the migratory sea turtle, Chelonia mydas, can smell a variety of chemicals 
dissolved in water in moderately low concentrations. Such detection is a pre- 
requisite sensory capability if, as has been suggested, chemical cues borne in ocean 
currents play a role in navigation. 



We thank Drs. A. Carr, ]. E. Bardach, D. R. Griffin and J. A. Nevin for 
encouragement and advice. The Caribbean Conservation Corporation aided us in 
the collection of green turtle eggs. This work was supported in part by Barnard 



194 M. MANTON, A. KARR, AND D. W. EHRENFELI) 

College faculty research grants and a grant from the Penrose Fund of the American 
Philosophical Society (to D. W. E. I. 

SUMMARY AND CONCLUSIONS 

1. The ability of the green turtle (Clicloiiia inydas) to detect various chemical 
substances dissolved in water has been investigated using operant conditioning 
techniques. The turtles pressed underwater keys to obtain food reinforcement 
in the presence of a chemical stimulus. 

2. The turtles were capable of underwater chemoreception of /?-phenethylalcohol, 
iso-pentyl acetate, triethylamine and cinnamaldehyde at approximate concentrations 
of 5 >! 10 " M or 5 : 10~ r> M, but not of L-serine or glycine at an approximate 
concentration of 10 ' M. 

3. Stimulus generalization occurred when turtles were shifted from one test 
chemical to another. 

4. Intranasal injection of 0.35 M zinc sulfate solution interrupted olfaction for 
periods of from 1 to 5 days. Treatment with 0.35 M saline or magnesium sulfate 
had no effect on the performance of the chemical discrimination. It was con- 
cluded on the basis of these experiments that chemoreception in Chclonia is largely 
or entirely mediated by olfaction rather than by taste. 

5. The advantages of the zinc-induced anosmia over surgical techniques and the 
possible use of the zinc treatment in field studies of orientation are discussed. 

6. Our results provide evidence to support the current theory that soluble com- 
pounds entering ocean currents from the vicinity of nesting sites might be de- 
tected by green turtles, and that this could aid in navigation. 

LITERATURE CITED 

ALBERTS, J. R., AND B. G. GALEF, JR., 1971. Acute anosmia in the rat: a behavioral test of 

peripherally induced olfactory deficit. Physio I. Behav.. 6 : 619-621. 
AZRIN, N. H., AND W. C. HOLZ, 1966. Punishment, Pages 380-447 in W. K. Honig, Ed., 

Operant Behavior : Areas of Research and Application. Appleton-Century-Crofts, New 

York. 
BOYCOTT, B. B., AND R. W. GUILLERY, 1962. Olfactory and visual learning in the red-eared 

terrapin, I'sciideinys scripta clci/ans Wied. /. E.rp. BioL, 39: 567-577. 
CARK, A., 1967. Adaptive aspects of the scheduled travel of Chclonia, Pages 35-55 in R. M. 

Storm, Ed., Animal Orientation and Navigation. Oregon State University Press, 

Corvallis. 
CARR, A., 1972. The case for long-range chemoreceptive piloting in Chclonia. Pages 469-483 

in S. R. Caller, ct a!. Eds., Animal Orientation and Navigation. NASA, Washington, 

D. C. 
EHRENFELD, D. W., AND A. L. KOCH, 1967. Visual accommodation in the green turtle. Science, 

155: 827-828. 
EVANS, W. E., AND J. BASTIAN, 1969. Marine mammal communication: social and ecological 

factors, Pages 462-470 in H. T. Anderson, Ed., Biology of Marine Mammals. 

Academic Press, Xevv York. 
IDLER, D. R., U. FAGERLUND AND H. MAYOH, 1956. Olfactory perception in migrating salmon 

I. L-serine, a salmon repellent in mammalian skin. /. Gen. Physio!., 39: 889-892. 
KOCH, A. L., A. CARR AND D. W. EHRENFELD, 1969. The problem of open-sea navigation: 

The migration of the green turtle to Ascension Island. /. TJicor. BioL. 22: 163-179. 



CHEMORECEPTION IN SEA TURTLE 

MANTON, M., A. KARR AND D. W. EHRENFELD, in press. An operant method for the study 
of sensory discriminations in the green trutle, Chclonia tnydas. Brain- Behav. Evol. 

NEVIN, J. A., 1970. On differential stimulation and differential reinforcement, Pages 401-423 
in W. C. Stebbins, Ed., Animal Psychophysics. Appleton-Century-Crofts, New York. 

PAPEZ, J. W., 1961. Comparative Neurology: A Manual and Text fur the Study of the Ncr- 
1'iins System of Vertebrates. Hafner, New York, 518 pp. 

PARSONS, T. S., 1967. Evolution of the nasal structure in the lower tetrapods. Amcr. Zool.. 
7: 397-413. 

TEICHMANN, H., 1959. Uber die Leistung des Geruchssinnes beim Aal, .-liif/uilla anuuilla L. 
Z. l'er</l. I'hysiol.. 42 : 206-254. 

TUCKER, D., 1963. Physical variables in the olfactory stimulation process. /. Gen. Phvsiol., 
46 : 453-489. 

TUCKER, D., AND T. SHIBUYA, 1965. A physiologic and pharmacologic study of olfactory re- 
ceptors. Cold Spring Harbor Symp. Quant. Bio!., 30 : 207-215. 

WALKER, W. F., 1959. Closure of the nostrils in the Atlantic loggerhead and other sea turtles. 
Coficia. 1959 : 257-259. 



Reference: Biol. Bull.. 143: 196-206. (August. 1972) 



ORCADIAN RHYTHMS : MECHANISM OF LUCIFERASE 
ACTIVITY CHANGES IN GONYAULAX 

LAUKA ML-MURRY AND j. w. HASTINGS 

Binlof/ical Laboratories, Harvard University, 16 Divinity Avenue, 
Cambridge, Massachusetts 02138 

Gon\aula.\' polyedra is a bioluminescent dinoflagellate which exhibits a daily 
rhythmic fluctuation in its capacity for hioluminescence (Hastings and Sweeney, 
1958). When grown using a LD 12:12 cycle (12 hours light alternating with 
12 hours darkness), the period of the hioluminescence rhythm is exactly 24 hours; 
the maximum luminescence is in the middle of the dark period. However, rhyth- 
micity will persist in constant laboratory conditions, where it assumes its "cir- 
cadian" period, close to hut not exactly 24 hours. Under these conditions the 
phase of the rhythm is independent of that of the earth's daily cycle (Aschoff, 
1956; Biinning, 1967). 

Little is known about the mechanism of circadian rhythms or about the bio- 
chemistry involved in their expression. 

In (lOnyaula.v activity of the extractable soluble enzyme luciferase displays a 
circadian rhythm which correlates well with the bioluminescence rhythm of the 
intact cell. Luciferase, which catalyzes in vitro luminescence via oxidation of a 
low molecular weight substrate "Gonyaulax luciferin," has been partly purified 
and characterized (Bode and Hastings, 1963; Fogel and Hastings, 1971). The 
luciferase activity in the supernatant resulting from centrifuging a cell homogenate 
for about 30 minutes at 27,000 X g fluctuates rhythmically with time of extraction, 
both for cells from a light-dark cycle (Hastings and Bode, 1962) and for cells 
from constant dim light (Fig. 1). Hastings and Bode (1962) reported that this 
rhythm was not one of total protein extractability and that the day-night difference 
in luciferase activity per mg protein was retained after an 8-fold purification. 

A number of other cases in which enzymatic activity varies from night to day 
are known (Sanwal and Krishnan, 1960; Potter, Gebert, Pitot, Peraino, Lamar, 
Lesher, and Morris, 1966; Rapoport, 1966; Given, Ulrich, Trimmer, and Brown, 
1967; Hardeland, 1969; Sweeney, 1969), but in only a few cases (Hardeland, 
Sweeney) has the molecular basis been examined, and in no case has it been well 
defined. 

We report here further experiments to discover the immediate biochemical basis 
for the cycling in Gonyaulax luciferase activity. We also discuss the contribution 
of the luciferase rhythm to the bioluminescence rhythm of the intact cell. 

MATERIALS AND METHODS 

Gonyaulax polycdra is a photosynthetic, bioluminescent, armored marine dino- 
flagellate about 40 /A in diameter. Two strains were used. The non-axenic 
strain was that used by DeSa (1964) and reported by him to have been isolated in 

196 



ORCADIAN LUCIFERASE RHYTHMS 



197 



b 

X 

3co 

el b 

U X 

_l {_, 

2 LJ 

g |l 

z z 

o o 



luciferase 



luminescence 
capacity ? ; 






TJ - 



70 80 90 100 110 120 

HOURS IN CONSTANT DIM LIGHT 



130 



FIGURE 1. Luciferase activity rhythm and luminescence capacity rhythm in constant light 
(160 footcandles), 22 C. The culture was transferred into constant light at the end of a light 
period. Cells were pelleted from 80 nil of culture by a 1.5 minute centrifugation at speed 3 in 
an International clinical centrifuge (more convenient for harvesting small volumes of culture 
than filtration through a Biichner funnel), suspended in 16 ml 0.05 M tris, 0.01 M EDTA, pH 8, 
0.005 M DTT, and homogenized once. The cell debris was removed by 5 minutes' centrifuga- 
tion at 2000 X g and reextracted with 8 ml. The first supernatant and second homogenate were 
combined and centrifuged 15 ruin at 27,000 X g. The supernatant was assayed for luciferase in 
assay mixture #1. 

1952 by Dr. B. M. Sweeney. Cultures were maintained as previously described 
(Fogel and Hastings, 1971). Tbe second strain (used only for the experiment 
shown in Figure 1) was an axenic clone derived in the laboratory of Dr. Robert 
Guillard, Woods Hole Oceanographic Institute, from a strain isolated by Dr. 
Sweeney in 1960. The data in Figure 1 accompanied other measurements, not 
pertinent here, which required an axenic culture. The second strain was main- 
tained in "f/2" medium ("f/2" medium is the "f" medium of Guillard and Ryther 
(1962) diluted in half with sea water). Both strains have similar luciferase 
rhythms. 

Luminescence was measured by a photomultiplier and amplifier as described 
by DeSa (1964). The instrument was calibrated at 490 nm using the secondary 
luminescence standards of Hastings and Weber (1963). The intensity of fluores- 
cent lights used in growing cells was measured with a Weston illumination meter 
model 756, quartz filter. 

Under LD 12:12 growth conditions, we found the luciferase activity of "mid- 
night" extracts (made after 5 to 7 hours of darkness) to be about 10 times that 



198 LAURA McMURKV AND J. W. HASTINGS 

of "mid-day" extracts (made after 5 to ',' hours of light), while under constant 
light the rhythm had a smaller amplitude (Fig. I). Therefore we used LD condi- 
tions and mid-day and mid-night extraction times to assure both a large dif- 
ference between minimum and maximum luciferase activities and predictability for 
the phase of the rhythm. A culture of 3000 to 10,000 cells/ml (uniform in any 
given experiment) was harvested by nitration on a Biichner funnel (except in 
Fig. 1) and the cells were suspended in either cold pH 6.0 extraction buffer 
(0.05 M sodium potassium phosphate) or cold pH 8.0 extraction buffer (0.05 M tris. 
0.01 M EDTA) with 0.001 to 0.005 M DTT. Extraction in the first buffer yields 
luciferase of approximately 35,000 molecular weight, the second 150,000 M.W. 
( Fogel and Hastings. l c) 71 ). Ten ml or more of extraction buffer was used per 
800 ml of culture. The cell suspension was passed twice through a stainless steel 
hand emulsiner (Fisher Scientific Co., catalog #11-504-2000). The homogenate 
was centrifuged in the HH4 swinging bucket rotor in a Sorvall refrigerated centri- 
fuge as described in the figure legends and the pellet discarded. 

In a study of luciferase extractability, guanidine was used in the extraction 
medium. Cells were extracted by homogenization in 5 M guanidine hydrochloride, 
0.005 M DTT, pi 1 6.7 at 22 C. As the control, cells were extracted "with the pH 
extraction buffer at 4 C. "Day" cells were harvested 5.5 hours after lights on 
and "night" cells 4.5 hours after lights off. The crude homogenate was centri- 
fuged at 25,000 X </ for 10 minutes to give a supernatant. The luciferase, denatured 
by guanidine, was renatured by 1/25 dilution into 0.05 M tris, 0.01 M EDTA, 
0.001 M DTT, 0.1 mg/ml HSA. pi 1 8.0 at 4 C. where complete recovery took about 
5 hours. Control samples were similarly diluted. Assays were done in assay 
mixture #2. 

To obtain luciferin for the luciferase assay, cultures from the day, or from 
the night after an hour's exposure to bright light (Bode, DeSa and Hastings, 
1 ( )(3), were harvested as decribed above and cells suspended in buffer in a boiling 
water bath. The buffer was 0.0025 M tris, 0.0005 M EDTA, pH 8.0; about 1.5 ml 
per flask of culture (800 ml) was used. After 2 minutes, the solution was chilled, 
made 0.005 M in DTT, centrifuged 30 minutes at 27,000 X g, and the supernatant 
frozen and stored in 1 ml portions at 57 C. 

Two different reaction mixtures were used for the luciferase assay, using a 
volume of 2 ml in both cases. Assay mixture #1 (Bode and Hastings, 1963) 
was 1 M ammonium sulfate. 0.1 M tris-maleate, 0.4 mg/ml BSA, 0.0025 M EDTA, 
pH 6.4 to 6.7. Assay mixture #2 (Fogel and Hastings, 1971 ) was simpler and so 
preferred in later experiments; it was 0.2 M sodium phosphate, 0.1 mg/ml BSA, 
pH 6.2. The assay was carried out by adding ( in either order ) luciferin and 
luciferase in prompt sequence ; the reaction was initiated by the last addition. The 
intensity was recorded at a fixed time after initiation (about 10 sec) and was pro- 
portional to the luciferase concentration over the range assayed. Light emission 
without added luciferin was negligible. Unless otherwise specified, the assays 
were done in duplicate or triplicate with an average error of 11%. 

Luminescence capacity refers to the amount of light emitted by the intact cell 
when stimulated by mechanical or chemical means. In these experiments, two ml 
of cell culture was placed in a 20 ml vial above a photomultiplier and 1 ml 0.06 N 
acetic acid was injected into the vial. The burst of light thus elicited was inte- 



ORCADIAN LUCIFERASE RHYTHMS 199 

grated electronically for 5 sec, at which time emission was complete. Assays 
were done in triplicate and had an average error of 4 c /c. 

The activity of luciferases from "day" and "night" cells were compared after 
centrifugation in sucrose density gradients. Pig heart lactate dehydrogenase pur- 
chased from Sigma Chemical Corporation was used as a marker enzyme to control 
for variations in sedimentation velocity from one gradient to the next. One hun- 
dred p\ of luciferase from a pH 8 tris extraction was mixed with 10 /A lactate 
dehydrogenase (0.21 mg/ml) and layered on top of a 4.5 ml, 5-16% convex ex- 
ponential sucrose gradient, made with 0.05 M tris, 0.01 M EDTA, 0.005 M DTT, 
pi 1 8.0. Gradients were spun 23.9 hours at 37,400 rpm in a S\Y 39 rotor in a 
Beckman ultracentrifuge model L2. Gradient tubes were impaled on a syringe 
needle and fractions of 10 drops were collected in small iced tubes containing 
25 p\ 0.05 M tris, 0.01 M EDTA. 5% sucrose, 0.1 mg/ml HSA. Two luciferase 
assays were done on each fraction ; 50 /A of the fraction was used for each. Assay 
mixture #2 was used. To assay for lactate dehydrogenase, sodium pyruvate to 
make 0.00076 M and XADH to make 0.058 mg/ml were freshly added to 0.03 M 
Na.,HPO 4 , pH 7.4; then to 1 ml of this in a cuvette was added a 25 (A gradient 
sample. The contents of the cuvette were mixed and the change in optical density 
at 340 nm per minute was measured on the 0.1 slideware of a Cary 15 recording 
spect rophotometer. 

Abbreviations used are: BSA, bovine serum albumin; DTT, dithiothreitol ; 
EDTA, ethylenediaminetetraacetate ; LD 12:12, a light/dark cycle with 12 hours 
of light alternating with 12 hours of darkness; NADH, reduced nicotinamide 
adenine dinucleotide ; and tris. tris-( hydroxymethyl )-aminomethane. 

RESULTS 

A trivial explanation for the luciferase activity rhythm, namely, that total 
protein might manifest such a rhythm in extractability, had been stated to be untrue 
(Hastings and Bode, 1962). Any individual enzyme such as luciferase, however, 
might still be bound more securely than proteins generally during the day than 
during the night and so be retained better by the cell debris. We made a pre- 
liminary check for such selective retention by assaying the crude cell homogenate 
(which includes the cell debris) for luciferase in the standard luciferase assay. 
Extractions made in pH 6.0 phosphate buffer showed that there was 2 to 3 times 
more luciferase activity in a crude cell homogenate than in the supernatant after 
the cell debris had been removed by centrifugation. This difference, however, was 
found in extracts made during the night as well as during the day. These measure- 
ments therefore gave no evidence that selective retention of assayable luciferase by 
cell debris during the day explained the luciferase rhythm. 

The possibility remained that only a fraction (say 10%) of the luciferase in 
"day" cells was similar in extractability and activity to that in "night" cells while 
the bulk of luciferase in day cells was rendered both inextractable and inactive 
by its location within the cell. Were this so, the luciferase might become assay- 
able if it could be released from its location. Therefore we tried different mechan- 
ical methods of extraction as well as different extraction media in an effort to 
bring the activity of day extracts up to the level of that of night extracts. 



200 



LAURA McMURRV AND J. W. HASTINGS 



(a)Luciferase 



(b)Luminescence 
capacity 



00 



o 

CD 
CO 



C 
O 



P H 6.0 EXTRACT 
P H 8.0 EXTRACT 




NIGHT 




DAY 



40 80 

M! luciferase extract 

FIGURE 2. (a) Comparison of luciferase activity in extracts made in 0.05 M phosphate 
buffer, pH 6 ("pH 6 extract") and in 0.05 M tris, 0.01 M EDTA, pH 8 ("pH 8 extract") 
during both the day and the night. Fifteen hours before the first harvest, cultures of cells 
were combined and divided between two flasks. Extractions were made with 0.001 M DTT 
7 hours after lights off (NIGHT) and 3.5 hours after lights on (DAY), using one flask for 
each time, one half of the flask for each of the two extraction pH's. The extract was spun 
3 min at 2,000 X g to remove cell debris, then 15 min at 20,000 X g. Assay mixture #1 was 
used, final pH 6.7, one assay per point, (b) Luminescence capacity measured in the same ex- 
periment just prior to cell harvest. 



In all cases we failed to accomplish this. Typical day-night differences in luci- 
ferase activity were observed whether the extraction was done by stirring the cells 
in buffer or by emulsification with the Fisher emulsifier. A Ten-Broeck glass 
homogenizer was found to be only about half as effective as the emulsifier in releas- 
ing luciferase during both the clay and the night. In addition, we found similar 
activities in extracts made in phosphate buffer at pH 6 and in tris buffer \vith 
EDTA at pH 8 at any given time, while the day activity was still about 10% of 
the night activity (Fig. 2). These findings are also of interest because it had been 
shown that the extraction medium determines which of two molecular weight 
forms of luciferase would be obtained, 35,000 (phosphate buffer, pH 6) or 150,000 
(tris buffer with EDTA, pH 8) (Fogel and Hastings, 1971). 

Another variation in the extraction medium involved the use of guanidine, 
which disrupts noncovalent bonds (which might be responsible for holding day 
luciferase more firmly in the cell) (Tanford, Kawahara, Lapanje, Hooker, Zarlengo, 
Salahuddin, Aune, and Takagi, 1967). Extraction in the emulsifier with SM 
guanidine did not release proportionately more luciferase activity from "day" cells 
than it did from "night" cells in comparison to pH 6 phosphate extraction buffer. 
(See Methods for details. The luciferase extracted in guanidine is denatured but 
is restored to complete activity by subsequent dilution in tris buffer at pH 8.) 
Extraction in quanidine apparently detached all the assayable luciferase from the 
cell debris, since the same recoverable activity was found in the guanidine super- 
natant above the cell debris as was found in the guanidine (and phosphate) crude 
homogenates before the cell debris was removed by centrifugation. Extraction in 



ORCADIAN LUCIFERASE RHYTHMS 



201 



o 

X 3 

o 

CD 
^ 

O 2 

"c 

D 

Z3 

cr 







(a) Mixing of DAY 
and NIGHT 
extracts 




night extract 



-O.I ml each 
extract 



day extract 



(tf Dialysis of DAY extract 
vs. NIGHT extract 



quanta/sec, x 
O.I ml x IO" 9 

.5 I 



NIGHT 
DAY 

key; 






NON-DIALYZED (CON) 
DIALYZED 



.1 .2 .3 4 

ml luciferase extract 



.5 



FIGURE o. Two experiments testing for presence of an inhibitor or activator of luciferase 
in extracts made during the day and during the night. Five days before harvest time a flask 
from constant dim light was diluted with new medium ; half these cells were put on one LD 
12:12 cycle, the other half on the same cycle, 12 hours out of phase. Cells were harvested 
6 hours after lights on or lights off and extracted in 0.05 M tris, 0.01 M EDTA, pH 8.0 
< 0.005 M DTT). The homogenate was centrifuged 1 hour at 23,000 X r/. (a) Mixing aliquots 
of day and night extracts to check whether activities were additive. (0.5 x) ml extraction 
buffer and x ml luciferase extract were added to assay mixture #1 containing 50 /J.I luciferin. 
(b) Dialysis of day extract against night extract. 1.5 ml day extract was put inside a dialysis 
bag and dialyzed 13 hours at 4 against 3 ml night extract; activity changes were compared to 
those of undialysed extracts containing pieces of dialysis hag (controls) ; 0.1 ml luciferase was 
assayed. 



phosphate butter, on the other hand (as mentioned above), apparently detached only 
^ to ^ of the available luciferase activity. 

From these experiments there is no evidence that the luciferase rhythm is 
due to a rhythm in luciferase extractability. \Ye now turn our attention to the 
possible presence of activators or inhibitors in the extracts. 

Were an activator present in the night extract in a greater than stoichiometric 
amount, this extract would enhance the day extract's activity ; conversely, a day 
extract, if it contained an inhibitor, should reduce the activity of the night extract. 
The total activity of such mixtures of day and night extracts was, however, 
approximately the sum of that of the two constituents (Fig. 3a). Several other 
experiments confirmed this; the slight inhibition seen in Figure 3a fell within ex- 
perimental error. 

If such an activator or inhibitor were present but in only stoichiometric amounts, 
the activity of mixtures would be additive. If it were, however, not tightly bound, 



202 



I.AURA McMURRY AXD J. W. HASTINGS 



TABLE I 

Effect of ammonium sulfate precipitation upon the activity of litciferase from the day and from the night . 

(The same luciferase extract was used as for Figure 3. The supernatant from the I hour's 

centrifugation was made 70'', in ammonium sulfate over a 5 minute period and stirred 

then for 10 minutes at 4 C. Tlic luciferase was pelleted at 15,000 rpm in a Sorcall 

SS34 in 3<> / unites and resuspended in 1 ml extraction buffer. Ten /JL! 

of extract wiis assayed far luciferase in the manner of Figure 3, 

A..S. = ammonium sulfate precipitation) 



Time at which extract 
was made 


Total activity of extract: 
(quanta sec) 


' , Recovery 


K.'lore A.S. 


After A.S. 


Day 

Night 


12 X K) 9 
120 X Id 9 


6.7 X Id 9 

72 X Id 9 


56' ; 
60', 



it should be removable upon purification of the enzvme. However, the difference 
between day and night luciferase activity (for the 150,000 molecular weight form) 
persisted after dialysis (even when day luciferase was dialyzed against night luci- 
ferase (Fig. 3bi ). ammonium sulfate precipitation (Table I I and sucrose velocity 
gradient centrifugation ( Fig. 4 ) , each done on crude enzyme. Therefore, either 
no activator or inhibitor was present, or, if one was, it did not separate from its 
luciferase during these purification treatments. Separation might have failed to 
occur if the hypothetical inhibitor or activator were bound to the luciferase or if 
it behaved similarly to luciferase during purification. 



DISCUSSION 

Summarizing these experiments, the rhythm of luciferase activity in crude ex- 
tracts of Gon\aitla.\- cells from a LI) 12:12 cycle does not appear to result from a 
rhythm of luciferase extractability. However we cannot completely exclude this 
possibility (for example, during the day, luciferase might be covalently bound to 
the- cell debris and rendered inactive). Further, it does not appear to be caused 
by a rhythm in concentration of an activator or inhibitor molecule of any variety 
1 >rcsent in greater than stoichiometric amount; nor does it appear to be caused 
by a rhythm in dissociable activator or inhibitor molecule present in stoichiometric 
amounts unless such a molecule is similar to luciferase in its behavior upon dialysis, 
ammonium sulfate precipitation, and sucrose velocity gradient centrifugation. 

The results thus rule out several explanations for the rhythmic change in luci- 
ferase activity. Two possible explanations appear to remain. One is that there 
are simply more luciferase molecules present in night cells than in day cells. 
Such a situation would imply large scale dc noro synthesis and degradation proc- 
esses in vivo not associated with growth. A method similar to that of Filner and 
Varner (1967) involving heavy isotopes C 13 and N 15 and sucrose velocity gradients 
was used to investigate this possibility (AlcMurry, 1971) but the results were 
not conclusive; they suggested that there may not be sufficient luciferase synthesis 
to support a hypothesis of complete de noro synthesis from amino acids. 

A second explanation would involve a chemical moiety which attaches and 



ORCADIAN LUCIFERASE RHYTHMS 



10 



o 

x 



O 

m 

X 

(J 



NIGHT EXTRACT 



100% RECOVERY 




luciferase 

lactate / xx 
dehydrogenase/7 





371 DROPS - 



.05 



P 

b 



\ 

a 

"c 5 
o 

ZJ 

cr 



( 


^o DAY EXTRACT 

N 100% RECOVERY 

; o 

/ \ 

^ 376 DROPS 

, s~\ TOT4L ^- 

y ^^ 


) 100 200 

BOTTOM drops 



.05 



FIGI/HK 1. Behavior of lucif erase activity from a day and a night extract during sedi- 
mentation of the extracts through sucrose gradients, showing persistence of the difference be- 
tween day and night activities. Half of each of two 800 ml cultures was harvested 6 hours 
after lights on (DAY) and 4.5 hours after lights off (NIGHT), extracted in 0.05 M tris, 
0.01 M EOT A, pH 8.0. 0.005 M DTT, centrifuged 40 min at 25.000 X g, and stored at -57 C. 
Gradients were run as described under Methods. 



detaches, possibly covalently, to and from the "backbone" of the luciferase molecule 
during each circadian cycle. The presence of this moiety would alter the luciferase 
activity. Luciferase would thus occur in two forms, one more active than the other. 
In the absence of growth, the total number of luciferase "backbone" molecule? 
would remain constant. One of two opposite situations might obtain: (1) the 
moiety might cause a 10-fold change in activity ; every "day" luciferase molecule 
might then be 10% as active as every "night" molecule, in which case the active 
species would differ chemically from day to night; (2) the moiety might com- 
pletely activate or inactivate ; all of the molecules would be active at night while 
only 10% of the molecules would be active by day. In this case the active species 
would be the same from day to night. 



204 LAURA McMURRY AM) J. W. HASTINGS 

For either situation, the moiety could he of any size or nature compared to the 
luciferase backbone, with appropriate effects upon the separation of backbone from 
backbone-plus-moiety during purification (the moiety could even be a cellular 
organelle). The moiety itself could undergo a daily de novo synthesis and de- 
struction or it could he always present in the cell. The moiety could be a protein. 
It should be noted in this respect that an activity rhythm is seen for both the 150,000 
and 35,000 molecular weight forms of luciferase ( Fig. 2 ). 

If there is any difference in the molecular weight between active dav and night 
luciferase, it is not large ( Fig. 4 ) ; the somewhat smaller sedimentation velocity for 
day luciferase seems to be at least partly a function of its lower activity, for if night 
enzyme is diluted to comparable activity, it has a similarly smaller sedimentation 
velocity (McMurry, 1 L )71 ). 

The rhythm of luminescence capacity parallels that of soluble luciferase activity 
(for example, see Fig. 1 and 2 ). What is the significance of this fact? 

Luminescence capacity can be measured in two ways, by stimulating the cells 
with acid to emit a burst of light (as in this report), or by bubbling them with 
air to cause many individual bright flashes (Hastings and Sweeney, 195S). Values 
obtained with acid are two to three times higher than those obtained by bubbling, 
but the rhythms are otherwise apparently equivalent (McMurry. l c >71 ) ; we may 
therefore in this discussion talk about cell flashes. 

Over 95% of the light emitted during bubbling comes from cell flashes 
(McMurry, l e >71). However, luminescent particles, the scintillons (DeSa, Has- 
tings, and Yatter. 1963). rather than soluble luciferase, are believed to be respon- 
sible for cell flashes (Hastings. Yergin, and DeSa, 1966; Eckert and Reynolds, 1967; 
McMurry, 1971; Fogel and Hastings. 1972). Hence the question is likely one 
of the relationship between the soluble luciferase and the scintillons. 

Scintillons can utilize free luciferin and likely also contain luciferase (Fogel, 
1970; Fogel and Hastings, l c >72). The luciferase extracted in soluble form may 
be in equilibrium /;; I'ivo with luciferase on the scintillons, or it may be solubilized 
from scintillons during extraction. In either case, if the amount of light which 
scintillons emit /;/ rh'o were for some reason proportional to their luciferase 
activity content, the soluble luciferase activity would reflect the luminescence ca- 
pacity. 

This research was supported in part by a National Science Foundation Research 
Grant CA>> 1(>51_ 7 . 1 .aura McMurry held a USPH 1'redoctoral Traineeship. 

SUMMARY 

The bioluminescent marine dinoflagellate Gonyuulu.r polycdra manifests similar 
circadian rhythms of bioluminescence capacity and extractable luciferase activity, 
both with maxima during the night phase. The immediate biochemical basis of 
the luciferase rhythm wa> in\ instigated, with the following findings : 

(1) The rhythm was present no matter which of several mechanical extraction 
methods and extraction media (including 5 M guanidine) were employed. The 
rhythm was present even in a crude cell homogenate. Thus the rhythm is likely 
not one of extractibility unless luciferase is inactivated while being covalently 
bound to cell debris during day phase. 



ORCADIAN LUCIFERASE RHYTHMS 205 

(2) Mixing experiments, ammonium sulfate precipitation, dialysis, and su- 
crose velocity gradient centrifugation showed that no dissociable activator or in- 
hibitor of luciferase caused the rhythm. 

Two possible hypotheses remain untested: (a) the occurrence of dc novo luci- 
ferase synthesis and destruction, (b) the attachment (perhaps covalent) and de- 
tachment of an activity-modifying moiety. 

The luminescence capacity rhythm is primarily a rhythm of quantity of light 
from cell flashes. Cell flashes probably originate from extractable particles termed 
scintillons which flash during assay. The relationship of the luciferase rhythm 
to the luminescence capacity rhythm is discussed from this view. 

Note added in proof: For recent findings on the luminescence capacity rhythm 
see R. Christiansen and B. M. Sweeney, 1972. Sensitivity to stimulation, a com- 
ponent of the arcadian rhvthm in luminescence in Gonyaulax. Plant Pliysiol.. 49 : 
994-997. 

LITERATURE CITED 

ASCHOKF, J., 1965. Circtidinn Clocks. North-Holland Publishing Co., Amsterdam. 479 pp. 
BODE, V. C, AND J. W. HASTINGS, 1%3. The purification and properties of the bioluminescent 

system in Ganyditla.v polyedra. Arch, liiochcin. Biophys., 103: 488-499. 
BODE, V. C., RICHARD DESA AND J. W. HASTINGS, 1963. Daily rhythm of luciferin activity 

in Gonyaitln.v polyedni. Science. 141 : 913-915. 

BUNNING, E., 1967. The I'hysioloi/icul Cluck. Springer- Verlag New York, Inc., 167 pp. 
CIVEN, M., R. ULRICH, B. M. TRIMMER AND C. B. BROWN, 1967. Circadian rhythms of liver 

enzymes and their relationship to enzyme induction. Science, 157: 1563-1564. 
, R. J., 1964. The discovery, isolation, and partial characterization of a bioluminescent 

particle from the marine dinorlagellate Gonyaula.r polyedra. Ph.D. thesis, University 

of Illinois, 87 pp. 
, R. J., J. W. HASTINGS, AND A. E. VATTER, 1963. Luminescent "crystalline" particles: 

an organized subcellular bioluminescent system. Science, 141 : 1269-1270. 
ECKERT, R., AND G. T. REYNOLDS, 1967. The subcellular origin of bioluminescence in Noctihtca 

tniliaris. J. Gen. Pliysiol.. 50: 1429-1458. 
FILNER, P., AND J. E. VARNER, 1967. A test for de noi'o synthesis of enzymes: density labelling 

with H 3 O 1S of barley a-amylase induced by gibberellic acid. Proc. Nat. A cad. Sci., 

58: 1250-1526. 
EOGEL, M. M., 1970. The relationship between the soluble and particulate bioluminescence in 

extracts of the marine dinorlagellate, Gonyaula.r polyedra. Ph.D. thesis, University 

of Illinois, 108 pp. 

FOGEL, M. M., AND J. W. HASTINGS, 1971. A substrate-binding protein in the Gonymda.v bio- 
luminescence reaction. Arch. Biochcni. Biophys., 142: 310-321. 
FOGEL, M., AND J. W. HASTINGS, 1972. Bioluminescence : mechanism and mode of control of 

scintillon activity. Proc. Nat. Acad. Sci., 69 : 690-693. 
GUILLARD, R. R. L., AND J. H. RvTHER, 1962. Studies on marine planktonic diatoms. I. 

Cyclotella nana Hustedt, and Detonnla confcrvacca (Cleve) Gran. Can. J. Microbiol, 

8 : 229-239. 
HARDELAND, R., 1969. Circadiane Rhythmik und Regulation von Enzymen des Tryptophan- 

Stoffwechsels in Rattenleber und -niere. Z. Vcrgl. Pliysiol., 63: 119-136. 
HASTINGS, J. W., AND V. C. BODE, 1962. Biochemistry of rhythmic systems. Ann. New York 

Acad. Sci., 98 : 876-889. 
HASTINGS, J. W., AND B. M. SWEENEY, 1958. A persistent diurnal rhythm of luminescence in 

Gonymila.i- polyedra. Biol. Bull., 115 : 440-458. 



206 LAURA McMURRY AND J. W. HASTINGS 

HASTINGS, J. W., AND G. WEBEK, 1963. Total quantum flux of isotropic sources. /. Opt. 

Soc. Amer., 53: 1410-1415. 
HASTINGS, J. W., M. VERGIN AND R. J. DnSA, 1966. Scintillons : the biochemistry of dino- 

flagellate bioluminescence. Pages 301-329 in F. H. Johnson and Y. Haneda, Eds., 

Biolniiiiiicscciicc in Pwi/rcss. Princeton University Press, Princeton, New Jersey. 
McMuRRY, L. M., 1971. Studies on properties and biochemistry of circadian rhythms in the 

bioluminescent dinoflagellate, Gonyanla.r /><>/\v</n/. Ph.D. thesis, Harvard University, 

134 pp. 
POTTER, V. R., R. A. GEBERT, H. C. PITOT, C. PERAINO, C. LAMAR, JR., S. LESHER AND H. P. 

MORKIS, 1966. Systematic oscillations in metabolic activity in rat liver and in hepa- 

tomas. L Morris Heptoma No. 7793. Cancer Res., 26: 1547-1560. 
RAPOPORT, M. L, l%(i. Circadian rhythm for tryptophan pyrrolase activity and its circulating 

substrate. Science. 153 : 1642-1644. 
SANWAL, G. G., AND P. S. KRISHNAN, 1960. Diurnal variation in aldolase and phosphatase 

activity in tin- cactus plant. Nature, 188 : 664-665. 
SWEENEY, B. M., 1%9. Transducing mechanisms between circadian clock and overt rhythms 

in Gouyaitla.r. ( 'an. J. fiot.. 47 : 299-308. 
SWEENEY, B. M., AND J. W. HASTINGS, 1958. Rhythmic cell division in populations of 

Gnnymifii.v polycilra. J. Protuzunl., 5: 217-224. 
TANFORD, C., K. KAWAHARA, S. LAPANJE, T. M. HOOKER, JR., M. H. ZARLENGO, A. SALA- 

HUDDIN, K. C. AUNE AND T. TAKAGi, 1967. Proteins as random coils. III. Optical 

rotary dispersion in 6M guanidine hydrochloride. /. Aincr. Client. Sue., 89: 5023-5029. 



Reference: B'wl. Hull.. 143: 207-214. ('August, 1972) 



OBSERVATIONS AND EXPERIMENTS ON METHODS OF 

FERTILIZATION IN THE CHAETOGNATH 

SAG ITT A HISPID A 

M. R. REEVE AND M. A. WALTER 

Roscnsticl Scliin>l of M urine and Atmospheric Science. University of Miami. 
Ill ]\'ickenl><tcker Caitsc-a'ay, Miami. Florida 3314V 

For many years, the only laboratory-maintained chaetognath was Spadella 
cephaloptera, a member of the single benthic genus. LTntil the development of cul- 
ture ability with Sagitta hispidu (Reeve, 1970a), no extensive behavioral observa- 
tions were possible in the pelagic genera, which are generally considered to con- 
stitute the second most important group of marine macroplankton. The process by 
which fertilization occurs in Spadella. involving a reciprocal transference of sper- 
matophores between two individuals, has been extensively documented by Ghirar- 
delli (e.g., 1968). For the other genera, there exist scattered observations of a largely 
circumstantial nature which support the possibilities of both cross- and self- 
fertilization. These observations are reviewed in detail by Reeve and Cosper 
(1972) who noted that Hyman (1959) stated that "Self-fertilization is thus ap- 
parently the rule in Sagitta." (page 2 ( M although Alvarino (1965) concluded for 
chaetognaths as a whole that "... it is generally accepted that cross-fertilization 
bv copulation is the rule" (page 132). Since both reviewers indicated the 
dominance of the genus Sui/itta within the phylum, these opinions are essentially 
contradictory. Ghirardelli ( l'K>8) preferred to conclude that one method does not 
necessarily exclude the other, even in the same species. 

Foremost amongst the evidence for self-fertilization is the direct observation of 
the migration of spermatozoa from the seminal vesicle forward along the tail and 
into the seminal receptacle in isolated individuals of the species Sagitta setosa 
(Jagersten, 1940; Ghirardelli. 19(>8; Dallot, 1%8). The last author was able to 
show that this resulted in the laying of fertile eggs in some cases. The evidence 
supporting cross-fertilization is based on observations of objects considered to be 
spermatophores attached to specimens from plankton samples (e.g., Dallot, as 
reported to Ghirardelli, 1968; David. 1958) and the specialization of structure 
of seminal vesicles in some species, such as 6". bipnnctata where the seminal vesicle 
is surmounted by a small cup with "saw teeth" edges. Ghirardelli (1968) be- 
lieved such structures might serve as copulatory organs whose form fits the female 
genital orifice of that species only, preventing mating between different species. 
Further circumstantial evidence for cross-fertilization in pelagic chaetognaths occurs 
in the observation of Murikami (1959) concerning specimens of Sagitta crassa 
which were found adjacent to each other with heads oriented in the opposite direc- 
tion, presumably copulating. In addition, the fact that cross-fertilization is normal 
in the only species to be extensively studied alive (Spadella cephaloptera), and 
exclusive self-fertlization at least on an evolutionary time scale seems highly un- 
likely, suggests that extensive observation of pelagic species would be necessary 
before attempting to discount any possibility of cross-fertilization. 

207 



208 



M. R. REEVE AND M. A. WALTER 




FIGURE 1. 



FERTILIZATION IN SAG1TTA 209 

The observations and experiments reported below show that both self- and 
cross-fertilization can occur in the same species and provide some indication of 
their relative frequency. 

OBSERVATIONS AND RESULTS 

Sagitta hispida Conant occurs in Biscay ne Bay, adjacent to the laboratory, and 
can be maintained successfully in culture (Reeve, 1970a) by providing large 
aquaria, frequently changed, well aerated water and live zooplankton for food. 
Observations were made upon populations in rectangular acrylic or glass aquaria in 
volumes of 40 liters with a water depth exceeding 45 cm. Individuals and pairs 
were isolated in 250 ml containers. 

Cross-fertilisation 

In laboratory culture acts of cannibalism by Sagitta hispida are frequently 
observed, especially in older animals. Usually the attacker seizes the head of its 
victim by means of its spines or "jaws" (rather than another part of the body) 
and proceeds to ingest it whole. When both animals are mature and hence of 
similar size, several minutes may elapse during which the pair engages in violent 
swimming motions. Often the animals separate, and swim off apparently unharmed, 
bearing a small dark attachment on the side of their bodies. Microscopical exam- 
ination has shown this attachment to be a spermatophore packed with motile sperm. 

We have each seen the initiation of attack once. In each case, two animals 
made several upward darting movements in close proximity to each other before 
becoming attached. In the first case, spermatophore transference occurred and 
the animals separated some 90 seconds later. In the second case, they detached 
within a few seconds without any transference. Rapid darting movements, in 
which two animals may swim closely together from the bottom to the top of the 
water column (45 cm) and back again within 5 seconds, are seen mostly in popula- 
tions in which attachments are occurring. We suggest that it is a behavioral sequence 
associated with copulation. During the attachment phase of the encounter, the 

FIGURE 1. (A-C) The copulatory "dance." Three separate pairs of animals attached at 
their head ends engaged in characteristic violent movements during which spermatophores were 
exchanged. In C, each of the pair has gained a spermatophore. (D-G) External migration 
of sperm. This sequence taken of a single animal shows (D) the newly implanted sper- 
matophore from which sperm are beginning to move out, (E) a strong posteriorly directed 
stream moving along the lateral body wall immediately above the right anterior fin, (F) the 
arrival of the stream over the right gonopore where it begins to traverse the dorsal body wall 
towards the left gonopore until (G) accumulations of sperm build up over each gonopore. 
(H) The penetration of sperm through the left gonopore into the sperm pouch (which can 
be seen running anteriorly, exterior to the ovary containing eggs, by virtue of the sperm filling 
it.) Some sperm still remain outside of the body wall. (I) A spermatophore artificially 
attached on the dorsal body surface, which illustrates the migration of sperm streams to the 
lateral body wall on both sides rather than (as in Spadella) travelling along the dorsal body 
wall. (J) The remains of a spermatophore artificially attached immediately behind the head 
laterally on the collarette, streams from which have meandered in the region failing to estab- 
lish a gonopore-oriented direction to their flow. Subsequent to this photograph, they became 
diffuse and disappeared. The approximate length of these mature animals is 9 mm and width 
of the trunk region 0.5 mm. Abbreviations are : fa anterior fin ; fp posterior fin ; g gono- 
pore ; o ovary ; p seminal pouch ; s sperm ; sp spermatophore ; sv seminal vesicle. 



210 M. R. REEVE AND M. A. WALTER 

frequency and violence of the muscular movements of the partners is much more 
intense than in normal swimming movements. Attached head-on (Fig. 1 A-C), 
their bodies flex and twist in close proximity along their lengths as they move 
upwards in a spiralling motion followed by intervals of rest when they sink 
downwards. 

At least 50 such encounters have been observed. Usually the encounter ended 
with both animals gaining and losing a spermatophore (Fig. 1C). Only in situa- 
tions where a single spermatophore was exchanged could its origin be in no doubt 
although we assume that all transferences were from the other animal. The 
moment of transference was never seen (the swimming movement appeared blurred 
when photographed with a flash of 1/250 sec duration). The site of attachment 
was always on the lateral trunk wall, often between the anterior and posterior 
fins, which is about one-third of the total body length anterior to the seminal 
vesicles. Although sometimes it varied in either direction, it was not to be found 
on the tail or head, or mid-dorsally or ventrally, nor on "the dorsal median line 
directly behind the ciliary loop" as in Spadella (Ghirardelli, 1968). No phe- 
nomenon has ever been seen in our populations in which animals were oriented 
head-to-tail, as occurs in Spadella while one is attached to a substrate, or as 
Murikami saw in Sagitta crassa. On one occasion three animals were observed 
attached at their head ends, and on separating at least two had lost and received 
spermatophores. 

Immediately after receipt of a spermatophore the animal may be removed from 
the aquarium, placed in a small dish and viewed microscopically. Invariably, 
within the few seconds which elapse prior to examination, sperm are already 
streaming across the body surface (Fig. 1 D). Since no empty spermatophore 
is left behind, we believe that the "spermatophore" consists of the tightly packed 
sperms enveloped in a thin layer of material of presumably adhesive mucoid nature 
which disintegrates as the sperm stream through it. Ghirardelli (1968) refers to 
the phenomenon as the dissolving of the spermatophore. Although several streams 
radiate out in various directions at first (Fig. IE), only the stream which proceeds 
laterally in a posterior direction continues to move, and it does so until it reaches 
the female gonophore on that side (Fig. 1 F), w r hich is situated close to the junc- 
tion of the trunk and tail. Some of the sperm then travel around the circumference 
of the body to the gonopore on the opposite side, so that both pores have accumula- 
tions of sperm over them (Fig. 1 G). The sperm enter and migrate along the 
seminal receptacle or pouch which extends along the outer edge of the ovary 
(Fig. 1 H). The process by which internal fertilization of the eggs then occurs 
is described in detail by Ghirardelli (1968) who provided a series of photographs 
showing a pattern of sperm migration from the dorsal body wall behind the 
ciliary loop in Spadella. In this case, the sperm path bifurcates to reach both 
gonopores at the same time. In the series of photographs in Figure 1 D-G, the 
elapsed time from spermatophore attachment at the level of the anterior end of 
the anterior fin to entrance at the gonopores was 10 minutes. The rest of the 
sperm which had not reached the gonopores quickly lost motility and are pre- 
sumably detached during swimming movements of the animal, because after a 
further 10 minutes there are none remaining on the outside of the body. This 
condition varies from Spadella, where nearly all the sperm travel to and enter 
the gonopores. 



FERTILIZATION IN SAGITTA 211 

Self-fertilization 

In the course of experimental work involving the measurement of mature live 
animals over a long period of time, five individuals were accidentally observed 
where sperm were emerging from a small rupture of the seminal vesicle anteriorly 
and migrating along the lateral edge of the tail, entering the gonopore on that 
side. Relative to the volume of sperm in the seminal vesicle, the sperm were very 
few in number. The animals subsequently died before laying eggs. 

Self-insemination can be artificially induced by breaking open a full seminal 
vesicle using a very fine needle which causes the spermatophore to "pop" out. 
By confining the animal to a small dish in which the depth of water is only 2-4 
mm, the spermatophore can usually be maneuvered to touch the body of the animal 
where it sometimes adheres. When placed on the lateral trunk wall, the sperm 
stream out according to the sequence of events described in the previous section. 
This procedure was performed only on animals which had been isolated before 
becoming mature, at a stage when seminal vesicles had not begun to develop, 
ovaries were tiny and contained only stage I eggs (both conditions which are 
known not to be reverted to after maturity in this species, Reeve, 1970b) and 
which were smaller than any mature individuals. This was to insure that there 
was no possibility that prior insemination could have taken place. A total of 41 
animals which were inseminated by their own sperm in this manner laid eggs which 
developed into larvae. None laid infertile eggs. Most of the animals which did not 
survive to lay eggs died during or within hours of handling, many obviously dam- 
aged in the process. 

In the preceding experiments, spermatophore adhesion was directed to the 
region where it occurs naturally in cross-fertilization. Some attempts were also 
made to place spermatophores on other parts of the body. Anterior to the ventral 
ganglion, including the region of the ciliary loop, sperm trails issued from the 
spermatophore in various directions but none travelled far back towards the gono- 
pore (Fig. 1-J). Results \vere less clear when spermatophores were placed on 
the tail. No strong directional flows occurred, but the close proximity of the 
gonopore and the flicking of the tail fin during swimming attempts were both 
probably factors in permitting a few sperm to occasionally reach the gonopores. 
It was difficult to obtain adhesion of spermatophores dorsally or ventrally, and 
where this occurred, as in the dorso-lateral position of Figure 1-1, sperm trails 
would first orient themselves laterally often on both sides of the body before 
moving towards the gonopore. Unlike the condition in Spadclla, therefore, sperm 
could not be induced to move down the mid-dorsal line of the body wall. 

Relative frequency of self- and cross-fertilisation 

It was reported (Reeve, 1970b) that larvae could result from eggs laid by 
isolated individuals. Such occurrences are infrequent but since that study was 
concerned with estimates of fecundity based on egg-laying, quantitative data on 
fertility were not obtained. The maturation and laying of eggs in Sagitta liispida 
is not dependent on prior insemination and fertilization. 

In a new series of experiments, 66 animals out of 86 isolated from immaturity 
as described above laid one or more batches of eggs amounting to a total of 186 
batches. Only four batches (2%} resulted in the production of larvae which 



212 M. R. REEVE AND M. A. WALTER 

themselves amounted to less than 17% of the number of eggs laid in those four 
batches. No batches were discarded until beyond the normal period required for 
hatching (36-48 hr) although fertile eggs would cleave within 10 minutes of being 
laid. 

Since it is possible that the confinement of a normally planktonic 9-mm long 
animal in 250 ml could adversely influence its ability to produce offspring, a pre- 
liminary experiment was run in which 10 pairs of immature animals were utilized. 
Only three pairs survived long enough to produce a batch of eggs, all of which 
were fertile. This experiment was not expanded because of mortality problems 
probably aggravated by cannibalism. The following experiment, however, demon- 
strated the fact that larvae could be produced in close confinement. 

This experiment was performed primarily to ascertain whether an animal 
once inseminated could store sperm for fertilization of subsequent batches of eggs. 
This ability is possessed by planktonic marine copepods (e.g., Marshall and Orr, 
1955). Observations suggested that sperm storage was unlikely because there were 
no visible signs of sperm in the seminal pouches on the day following insemination 
(using Xl2 magnification). Initially, their position can be seen even by the 
unaided eye. 

Mature animals with well-developed ovaries were removed from aquarium 
populations and isolated. Of 32 animals isolated, 24 subsequently laid a batch of 
eggs, of which 10 were fertile and resulted in larvae only slightly fewer in num- 
ber than eggs laid. A total of 23 further batches were laid by this group, all 
of which were infertile. In the case of those whose first batch was infertile, we 
presume that they had been removed from the population before insemination had 
occurred. There was no evidence that insemination conferred fertility on any but a 
single batch of eggs. 

DISCUSSION 

On the basis of our own observations of Sagitta hispida, we would agree with 
Alvarino that cross-fertilization by copulation is the rule. Strictly, most observa- 
tions in the literature have concerned insemination rather than fertilization. Our 
indication of successful fertilization was the hatching of a larva, but we have no 
information on the viability or ultimate fecundity of such larvae. In Sayitta 
hispida our observations suggest that larvae are normally produced following in- 
semination during copulation which can sometimes be proved to be cross-in- 
semination (where only a single spermatophore is involved). Self-insemination 
is rare but has been shown to occur, and can readily be artificially induced to pro- 
duce fertile eggs. 

It might be argued that transference of a spermatophore from the seminal 
vesicle to the trunk of the same individual might occur during copulatory activities, 
whether accidentally or not. The chances of the seminal vesicle coming into con- 
tact with another part of its own body would seem much lower than of touching 
the other animal. On one occasion only, a single mature individual in an aquarium 
was seen to be violently twisting its body as in the copulatory sequence. There 
was, however, no spermatophore relocation. 

The obvious method by which two planktonic chaetognaths could become 
attached for copulation is by use of their grasping spines, since their other append- 



FERTILIZATION IN SAG1TTA 213 

ages, the fins, contain no musculature (Ghirardelli, 1968). We do not know 
whether an act of cannibalism is behaviorally different from copulation. When 
an individual seizes an animal smaller than itself, the wider spread of its spines 
enables it to encircle and render ineffective the spines of the smaller animal. 
When animals are of similar size, as two mature animals would be, it would be 
much more difficult for one to gain the advantage. In the ensuing struggle, 
spermatophores may be transferred and the animals may separate because neither 
can get a decisive grip on the other. In support of this possibility that copulation 
and attempted cannibalism are different results of the same process, we have occa- 
sionally seen mature animals in aquaria which have just ingested another chaeto- 
gnath which appeared to be of similar size, where the attacker bore a spermatophore 
on its body. This strongly suggested that both copulation and cannibalism had 
occurred. 

Reeve (1966) noted that he had never observed cannibalism in Spadclla 
cephaloptera and this observation still holds true in observations of the breeding 
population which we maintain in this laboratory. He suggested that the benthic 
Spadella might have developed a mechanism to prevent self-predation based on 
recognition of different kinds of swimming vibrations, since it congregated on sur- 
faces rather than being dispersed in a 3-dimensional space where there was less 
chance of encountering a member of the same species rather than a food organism 
such as a copepod. Since copulation in Spadella is achieved with the aid of 
adhesion of one of the pair to the substrate, the mechanisms of cannibalism are not 
necessary for copulation. 

The distinction between Spadella and the planktonic chaetognaths is not ab- 
solute. Sagitta hispida, S. helenae and possibly other neritic species possess the 
ability to attach to surfaces such as the aquarium wall. Unlike Spadella, however, 
where the newly hatched larvae attach on hatching and 95% of the population 
is usually attached at any one time. Sagitta hispida does not attach significantly 
until approaching maturity and even then most of the population is usually to 
be found swimming in the water column, in aquaria. Egg masses on aquarium 
walls indicate that 6". liispida utilizes surfaces for egg-laying, but we have not as 
yet seen indications that this species can transfer spermatophores while attached 
to a surface. 

Ghirardelli (1968) was also able to induce self-insemination in Spadella 
cephaloptera although he had no evidence that it could occur unaided, and his ob- 
servations of sperm migation in this region suggested that it was unlikely. Our 
observations suggest also that it is unlikely, but that it can occur. In Sagitta 
setosa, on the other hand, Ballot (1968) maintained isolated individuals in which 
50% of the spawnings gave fertile eggs. He did not observe any phenomena 
indicative of cross-fertilization, although he did not discount its existence in that 
species. 

Since Sagitta hispida is the only planktonic species currently being routinely 
raised over its whole life cycle in the laboratory, it is clearly too early to make 
generalizations covering the entire range of planktonic Chaetognatha with respect 
to relative frequency of cross- and self-fertilization. If. indeed, self-fertilization 
is a regular phenomenon which becomes more likely when opportunities for cross- 
fertilization are minimal, it might be expected to occur more frequently in species 
habitually in lower densities such as typically oceanic and bathypelagic ones. 



214 M. R. REEVE AND M. A. WALTER 

This work was supported by NSF Contract No. GA 28522X. Our thanks are 
due Drs. Harding Owre and Mary Rice for their interest, advice and reading of 
the manuscript. 

SUMMARY 

1. The first description is provided of the act of copulation of a planktonic 
chaetognath which leads to successful insemination and fertilization. It consists 
of a behavioral sequence in which partners maintain contact at their head ends 
while engaging in violent movements which result in the transfer of spermatophores. 

2. Acts of self-insemination in the same species are also described, as \vell as 
experiments in which self-insemination and successful fertilization are induced. 

3. The consequences of attachment of spermatophores to various parts of the 
body are described and compared to observations on the benthic genus Spadclla. 

4. Experiment > .showed 2% of batches of eggs laid by individuals isolated 
prior to maturity produced hatchings, suggesting that self-fertilization, although 
uncommon, does occur naturally. 

5. Experiments indicated that one insemination was effective for only a single 
batch of eggs. 

6. The similarities between copulation in Spadclla and Sagitta are discussed 
and the possibility that self-fertilization presents a short-term survival mechanism 
for populations at low density is suggested. 

LITERATURE CITED 

ALVARINO, A., 1965. Chaetognaths. Oceanogr. Mar. Biol. Ann. Rev., 3 : 115-194. 

DALLOT, S., 1968. Observations preliminaires sur la reproduction en elevage du Chaetognathe 

planktonique Sagitta sctosa Miiller. Rapp. Commn. Int. Mcr. Medit., 19: 521-523. 
DAVID, P. M., 1958. A new species of Eukrolmia from the Southern Ocean with a note on 

fertilization. Proc. Zool. Soc. London, 131 : 597-606. 
GHIRARDELLI, E., 1968. Some aspects of the biology of chaetognaths. Adran. Mar. Biol., 6 : 

271-375. 

HYMAN, L. H., 1959. The Invertebrates: Volume 5. Smaller Coelomate Groups. McGraw- 
Hill Book Company, New York, 783 pp. 
JAGERSTEN, G., 1940. Zur Kenntnis der Physiologic der Zeugung bei Sagitta. Zool. Bidr. 

Uppsala, 18 : 397-413. 
MARSHALL, S. M., AND A. P. ORR, 1955. The Biology of a Marine Copcpod Calanus fin- 

marchicus (Gunncrus}. Oliver and Boyd, Edinburgh, 188 pp. [Reprint of 1st Edition; 

1972, Springer-Verlag, New York, 205 pp.] 
MuuiKAMr, A., 1959. Marine biological study on the planktonic chaetognaths in the Seto 

Inland Sea. Bull. Naikai Reg. Fish. Res. Lab., 12 : 1-186 [cnglish summary 1. 
REEVE, M. R., 1966. Observations on the biology of a chaetognath. Pages 613-630 in H. 

Barnes, Ed., Some Contemporary Studies in Marine Science. Allen and Umvin, 

London. 
REEVE, M. R., 1970a. Complete cycle of development of a pelagic chaetognath in culture. 

Nature, 227: 381. 
REEVE, M. R., 1970b. The biology of Chaetognatha. I. Quantitative aspects of growth and 

egg production in Sagitta hispida. Pages 168-189 in J. H. Steele, Ed., Marine Food 

Chains. Oliver and Boyd, Edinburgh. 
REEVE, M. R., AND T. C. COSPER, 1972. Chaetognatha. In A. C. Giese and J. S. Pearse, Eds., 

Reproduction of Marine Invertebrates Volume 2 (in press). Academic Press, New 

York. 



Reference : Biol. Bull., 143 : 215-221. (August, 1972) 



EXCYSTATION OF THE APOSTOMATOUS CILIATE, HYALOPHYSA 
CHATTONI, WITHOUT METAMORPHOSIS 1 

RUTH VANSTORY SCHAUER 
North Carolina State University at Raleigh, Raleigh, North Carolina 27607 

Apostome ciliates are found encysted on the exoskeletons of Crustacea, excyst- 
ing at the molt of their host (Chatton and Lwoff, 1935). A major group, the 
exuviotrophs, excyst and feed only on the fluid from the host's molt ; while another 
large group, the histotrophs, also excyst at the death of an injured host and feed 
on the released body fluids. In either case the excystation of the encysted form 
(phoront) is preceded by an extensive metamorphosis. In the histotrophs the 
metamorphosis occurs soon after the migratory form settles on the host, but exuvio- 
trophs metamorphose immediately before the ecdysis of the host. The result of this 
metamorphosis is a feeding stage (trophont) capable of engorging up to 30 times 
its initial volume (Bradbury and Trager, 1967b). After this rapid feeding, the 
apostome encysts and divides into numerous small ciliates (tomites) which seek 
out and settle on a new host. 

Previous studies (Miyashita, 1933; Chatton and Lwoff, 1935; Trager, 1957; 
Bradbury and Trager, 1967b) suggest that the exuviotrophic phoronts are stim- 
ulated to metamorphose and excyst by the leakage of some compound or mixture 
of compounds that builds up in the host prior to ecdysis. Accordingly, the phoront 
stage of Hyalophysa chattoni found on brackish water shrimps was subjected to 
solutions of various substances that were likely to be concentrated in the shrimp 
just before ecdysis. 

MATERIALS AND METHODS 

Grass shrimps (Palaemonetcs pugio and P. intermedius) were collected near 
the Pamlico Sound Research Station, at Aurora, North Carolina. As many as 
several hundred were collected and held in two ten-gallon aquaria filled with 
13.3%o artificial sea water salts (Aquarium Systems Inc.), and equipped with 
aerater and filter. While the water temperature (22 to 25 C) and specific gravity 
(1.015) remained fairly constant, the pH varied between 6 and 8. The shrimps 
were fed daily. Of the 298 molting cycles recorded, 70% were 1 to 3 weeks long. 
All of the fresh molts contained Hyalophysa chattoni [identified by silver impregna- 
tion by the Chatton-Lwoff (Corliss, 1953) and the Protargol (Kirby, 1950) 
procedures]. 

Premolt shrimps were recognized by the gap that widens between the newly 
formed exoskeleton and the ends of the antennal scales and uropods as ecdysis 
nears (Passano, 1960). Metamorphosis in living Hyalophysa was recognized by 

1 This work is part of a thesis submitted to North Carolina State University, Raleigh, in 
partial fulfillment of the requirements for the M.S. degree in Zoology. 

215 



216 RUTH VAX STORY SCHAUER 

crowding of food plaquettes to one side and the mid-ventral position of the con- 
tractile vacuole (Bradbury and Trager, 1967a). 

Phoronts for in vitro experiments were obtained by cutting off a patch of exo- 
skeleton containing numerous phoronts. On the shrimp, Hyalophysa preferentially 
settles in the cuticular depression at the base of the eyestalk. The exoskeleton of 
the living shrimp was cut from the base of the rostrum, medial to the base of the 
eyestalk beyond the field of phoronts. The second cut from the ventral edge of the 
carapace extended dorsally to intersect the first cut. The final cut was made 
parallel to the body surface just under the exoskeleton severing the musculature 
and connective tissue. These three incisions released a fragment that included 
the anterior end of the gill chamber, the antennule, the antenna including the 
antennal scale, the eyestalk, and the base of the rostrum. 

The fragment was washed vigorously in brackish water (13.3%c artificial 
sea water with 500 units penicillin/ml and 0.05 mg streptomycin/ml (Bradbury 
and Trager, 1967b). The underlying muscle and connective tissue were picked 
away with forceps leaving only pieces of exoskeleton with the attached phoronts. 
During this half-hour procedure, the pieces were washed at frequent intervals to 
dilute the released body fluids. 

All experimental solutions were made from the antibiotic brackish-water solu- 
tion. All controls were in the antibiotic brackish water alone. 

The cleaned pieces of exoskeleton were placed in 1 ml samples of solution in 
clear plastic disposable depression dishes (Scientific Products) and the entire dish 
was covered with "Parafilm" (American Can Company). A typical experiment 
would include 4 replicates of the control and 4 replicates of the experimental solu- 
tion using phoronts from the same shrimp. The number of phoronts in each 
depression was counted and the percent of excystation was calculated by counting 
the remaining cysts at 4 hour intervals for 24 hours. 

To obtain body fluid from uninfected Palaemonetes, the shrimp was crushed in 
about 0.5 ml of the antibiotic brackish-water solution. The fluid was collected in a 
hypodermic syringe and mixed with equal parts of the antibiotic brackish water. 

All chemicals, except the ecdysterone, used to make experimental solutions 
were obtained from Sigma Chemical Company. The ecdysterone came from Mann 
Research Laboratories. 



RESULTS 

A surprising observation in the course of these experiments was that the 
excysting phoronts on intermolt shrimps resemble in all visible respects the normal 
migratory stage, the tomite (Bradbury, 1966). No compound used in these ex- 
periments triggered metamorphosis of phoronts on intermolt shrimps. The phoronts 
were tested for excystation from 2 to 12 days after settling on the shrimps. In 
all experimental io'lutions, including 0.05 M Tris-HCl buffer (pH 9.0), 0.5% 
(3-D glucose, 0.5% glycogen (from shellfish), 0.5% N-acetylglucosamine, and 
10' 7 M ecdysterone, the immerging ciliate had the body form and ciliary pattern 
of the tomite. 

While the phoronts on intermolt shrimps always excysted as tomites, those on 
permolt shrimps excysted either as tomites or trophonts. In premolt shrimps 



EXCYSTATION WITHOUT METAMORPHOSIS 



217 



TABLE I 

Averaged per cent excy station* of trophonts and tomites from replicate 
experiments using 4 individual premolt shrimps 



Solutions 


# 

Cysts 


Hours 


4 


8 


12 


16 


20 


24 


Control 


156 


51% 


58% 


83% 


87% 


Q7P/ 
0' /O 


87% 


I Glycogen (0.5)% 


309 


45 


75 


90 


96 


96 


96 


Control 


234 


21 


69 


90 


90 


90 


90 


II Glycogen (0.5%) 


200 


5 


82 


95 


96 


96 


96 


Control 


111 


13 


39 


97 


99 


99 


99 


ill Glucose (0.5%) 


182 


12 


22 


98 


100 


100 


100 


Ecdysterone 
















(10~ 7 M) 


118 


8 


23 


97 


100 


100 


100 


Control 


48 


45 


61 


82 


87 


87 


87 


IV Glucose (0.5%) 


71 


55 


77 


92 


47 


97 


97 


Glucosamine 
















(0.5%) 


88 


55 


69 


85 


91 


9.5 


93 



* Per cent excystation was calculated after counting the remaining cysts. Only trophonts 
were found swimming in the dishes after 4 hours. Both trophonts and tomites were found after 
8 hours. After 12 hours only tomites were found. 

(Table I) phoronts excysted as trophonts within 4 hours and continued to excyst 
as trophonts for another 4 hours. But after 8 hours, the remaining phoronts 
excysted as tomites. Excystation of tomites from intermolt shrimps (Table II) 
started after an initial lag of 4 hours and increased rapidly to 80% or higher by 
12 hours. Excystation was slower from 12 to 16 hours with little occurring 
after 16 hours. The general pattern of per cent excystation for both intermolt 
and premolt shrimps was the same after early differences. No significant effects 
on per cent excystation due to treatments alone were observed in any of the 
experiments. Transformation and analysis of variance of data for each shrimp were 
carried out using the Statistical Analysis System developed at NCSU for the use 
of the IBM 360/75 computer at Triangle Universities Computing Center. 

In light of these unexpected results and because experiments using blood have 
resulted in excystation of trophonts from other crustaceae (Miyashita, 1933; Chat- 
ton and Lwoff, 1935; Trager, 1957; Bradbury and Trager, 1967b), shrimp body 
fluid was used as an experimental solution, with intermolt shrimps. 

Experiment 1 

Three live infested shrimps were placed in body fluid from other shrimps diluted 
1 : 1 with antibiotic brackish water. No excystation was observed after 24 hours. 

Experiment 2 

Two fields of phoronts were cut from a shrimp 4 days from its last ecdysis. 
Neither piece was cleaned. One was placed in extra body fluids, while the other 



218 



RUTH VANSTORY SCHAUER 



was placed in the antibiotic brackish water alone. Excystation of tomites occurred 
from the first piece after 2 hours and from the second after 9 hours. 



Experiment 3 

One shrimp was found to have phoronts on the bristles of its maxillae. These 
phoronts excysted much more slowly than phoronts from the cuticular depression 
at the base of the eyestalk of the same shrimp. At 8 hours, 90% had excysted as 
tomites in the depression while none on the bristles excysted until after 8 hours 
(73% at 12 hours). 



Experiment 4 

When shrimps killed by stabbing near the field of phoronts were left in 
brackish water, no excystation of the phoronts occurred. Apparently they died 
on their hosts. 

To test whether the excysted tomites would behave as normal tomites, they 
were offered uninfested shrimps as hosts. The tomites darted about over the 
shrimp's body as normal tomites would, but none of the excysted tomites were ob- 
served to re-encyst. 



TABLE II 

Averaged per cent excystation of tomites from replicate experiments using 
6 individual intermolt shrimps 



Solutions 


# 
Cysts 


Hours 


4 


8 


12 


16 


20 


24 


Control 


50 





43% 


81% 


83% 


88% 


92% 


I Glycogen (0.5%) 


55 





57 


95 


100 


100 


100 


Control 


96 





94 


100 


100 


100 


100 


II Glucosamine (0.5%) 


108 





87 


96 


98 


98 


98 


Control 


61 





90 


97 


97 


97 


97 


III Glycogen (0.5%) 


33 





63 


97 


97 


97 


97 


pi I 9.0 (0.05 M Tris-HCl buffer) 


28 





100 


100 


100 


100 


100 


Control 


26 





29 


58 


69 


74 


84 


IV Glycogen (0.5%) 


20 





25 


50 


90 


90 


90 


pH 9.0 (0.05 M Tris-HCl buffer) 


32 





38 


66 


82 


82 


86 


Control 


120 





61 


85 


89 


91 


91 


V Glycogen (0.5%) 


76 





65 


86 


87 


92 


92 


pH 9.0 (0.05 M Tris-HCl buffer) 


71 





79 


87 


88 


88 


94 


Control 


121 





16 


83 


88 


91 


91 


VI pH 9.0 (0.05 M Tris-HCl buffer) 


79 





11 


63 


79 


82 


82 



EXCYSTATION WITHOUT METAMORPHOSIS 219 

DISCUSSION 

Because glycogen, glucose, and glucosamine markedly increase in Crustacea 
prior to ecdysis (Passano, 1960; Florkin, 1960), these compounds seemed likely 
to leak from the host just prior to the molt and perhaps thereby induce morpho- 
genesis in the exuviotrophic apostome. Glucose and gycogen were also tested in 
antibotic brackish water buffered at pH 9.0, the pH of molting fluid (Dennell, 
1960) . None of these substances effected metamorphosis of Hyalophysa on shrimp 
although the same species on the hermit crab has been reported to metamorphose 
in weak solutions of glycogen (Bradbury and Trager, 1967b). On the hermit 
crab, phoronts are found primarly between gill lamellae within the gill chamber 
(Bradbury, 1966). The phoronts found on hermit crabs never have been observed 
to excyst as tomites. In view of this as well as the results of Cleveland and Nutting 
(1955) with the flagellates of the wood roach, Trager (1957) has suggested that 
molting hormone might effect the metamorphosis of exuviotrophs. Accordingly 
ecdysterone was tested for its effect on excystation with no observable results. 
Perhaps, as Trager has suggested, a series of events must occur to induce meta- 
morphosis and excystation. 

Two variables in all the experiments could not be completely controlled : ( 1) the 
amounts of body fluid in contact with phoronts during dissection, and (2) the 
amount of tactile stimulation the cysts received. Although the pieces of exoskeleton 
were washed repeatedly with clean solutions during the cleaning of the pieces, 
the phoronts still were bathed in body fluids for as long as one half hour. Per- 
haps Experiment #3 with phoronts encysted on bristles was the only experiment 
in which these two factors might not be significant. Although the phoronts on 
the bristles excysted after a longer lag period and did not excyst in as great num- 
bers, excystation still occurred. 

It should also be considered that all the conditions of habitat provided by the 
shrimp were not duplicated in vitro. Living shrimps always have some part of 
their anterior body in constant motion. Antennae and antennal scales move, the 
scaphognathite beats continuously, the maxillipeds and mouth parts are usually in 
motion. These movements are in addition to the usual walking and swimming 
movements of the shrimp. The effect of such movements is to bathe the phoronts in 
a constant through variable stream of water. It would seem that such a constant 
flow of water would provide the phoronts with a good supply of oxygen. Since 
phoronts of all apostome species whose life cycle is known settle on well aerated 
sections of the exoskeleton, the phoronts may have a relatively high oxygen re- 
quirement. They do move within the cyst and their contractile vacuoles are 
active. None of these experiments tested the effect of the experimental substances 
moving over the surface of the body (i.e., phoronts bathed in a moving stream of 
a substance for a time before the current was stopped). 

Since phoronts die in situ on intermolt shrimps (dead from any cause), the 
loss of currents of water in addition to failure to release body fluids until de- 
composition begins may explain why these phoronts do not excyst. Another pos- 
sibility is that the lack of oxygen affects the phoronts before the body fluids leak 
from the shrimp. 

In Experiment #1 (living shrimps in 1:1 brackish water and body fluids) no 
excystation occurred even though the two conditions of water currents and body 



220 RUTH VANSTORY SCHAUER 

fluid were met. However, this experimental solution was cloudy and gummy with 
coagulated shrimp blood. Although antibiotics were added, bacterial action was 
just retarded not stopped. The viscous water currents were unlikely to be com- 
parable to water currents in the shrimp's natural environment. On the other hand, 
excystation may require both body fluids and cessation of movement. If this were 
so, the results of Experiments 2 and 3 would be expected because the large amount 
of body fluids in combination \vith a lack of water currents would lead to excysta- 
tion. In Experiment #4 the stabbed shrimp would release body fluids, but they 
would be quickly diluted. The wound was behind the field of phoronts. This 
would mean that the flow of water would carry the body fluids away from the 
phoronts. The swift coagulation of shrimp blood would quickly stop any great loss 
of fluid. Thus this experiment would have to be considered inconclusive. 

Since it was not feasible to give the cysts close microscopic scrutiny before 
removing them from the shrimp, it could not be ascertained whether metamorphosis 
of some had already occurred. Some premolt shrimps were taken very close to 
the oncoming molt. In these cases trophonts excysted during the dissections. It 
seems quite likely that metamorphosis had occurred before dissection was begun 
in such cases. It is also reasonable that the events leading to metamorphosis had 
already started for the other phoronts that excysted as trophonts. This excysta- 
tion of trophonts is common when dealing with premolt shrimps. The unexpected 
result was the reappearance of the tomites. 

However this reappearance of tomites does provide the evidence hitherto lack- 
ing for the independence of metamorphosis and excystation. Chatton and Lwoff 
(1935) imply from their discussions on metamorphosis of Synophrya that meta- 
morphosis and excystation are in fact two different processes. Phoronts of apos- 
tomes have never been shown to excyst as tomites at any time before. Therefore, 
the readiness of the phoront to excyst under stress, and the reappearance of the 
tomite from the phoretic cyst were completely unexpected. 

Even though the excysted tomites were not observed to settle on new hosts, 
their behavior was like that of tomites seeking a host in the normal course of their 
life cycle. Probably the tomite has only enough reserves to settle and encyst one 
time. Reserves remaining in the phoront are probably used for excystation. Since 
the tomite can not feed, its reappearance in this form is fatal to it. 

In the course of this work no phoronts have been seen on the molt of the shrimp. 
Nor were empty cysts observed on intermolt shrimps. Apparently the normal life 
cycle proceeds without accident unless experimentally altered. But the results of 
this series of experiments establish that the ability to excyst is maintained 
throughout the phoretic stage. 

I want to thank Dr. P. C. Bradbury the chairman of my graduate committee, 
and Drs. D. 1 Smith and D. Huisingh for their guidance and help. I am grateful 
to Dr. Larry Nelson and Mr. Frank Verlinden for their advice and assistance with 
the statistical analyses of my experiments. This work was supported in part by 
a grant from the National Institutes of Health (RR-00011~OQ) for Computer Use 
in Health Sciences. 

SUMMARY 
Pieces of exoskeleton bearing Hyalophysa phoronts from the bases of the eye- 



EXCY STATION WITHOUT METAMORPHOSIS 21 

stalks of intermolt and premolt Palacnionctcs were placed in antibiotic brackish- 
water solution containing 0.05 M Tris-HCl buffer at pH 9.0, 0.5% glycogen, 
0.5% /3-D glucose, 0.5% N-acetylglucosamine, and 10' 7 M ecdysterone. Controls 
were placed in antibiotic brackish water alone. In all experiments, the phoronts 
showed the same rates of excystation in control and experimental solutions. The 
phoronts excysted as tomites from intermolt shrimps, while both trophonts and 
tomites excysted from premolt shrimps. Experiments using body fluids from the 
host shrimp have indicated that the substance causing this unexpected excystation 
of tomites is in the body fluids of the host. 

The resulting excystation of tomites from the phoretic cysts of Hyalophysa 
establishes the separation of metamorphosis and excystation in apostome ciliates. 

LITERATURE CITED 

BRADBURY, P. C., 1966. The life cycle and morphology of the apostomatous ciliate, Hyalophysa 

chattoni n.g., n.sp. /. ProtozooL, 13 : 209-225. 
BRADBURY, P. C., AND W. TRACER, 1967a. Metamorphosis from the phoront to the trophont in 

Hyalophysa. J. ProtozooL, 14 : 307-312. 
BRADBURY, P. C, AND W. TRACER, 1967b. Excystation of apostome ciliates in relation to 

molting of their crustacean hosts. II. Effect of glycogen. Biol. Bull., 133: 310-316. 
CHATTON, E., AND A. LWOFF, 1935. Les cilies apostomes. I. Aperc.u historique et general : 

etude monographique des genres et des especes. Arch. /.ool. E.vp. Gen., 77 (Fasc. 1) : 

1-453. 
CLEVELAND, L. R., AND W. L. NUTTING, 1955. Suppression of sexual cycles and death of the 

protozoa of Cryptocercus resulting from change of hosts during molting period. 

J.Exp.Zool., 130: 485-513. 
CORLISS, J. O., 1953. Silver impregnation of ciliated protozoa by the Chatton-Lwoff technic. 

Stain Tech., 28 : 97-100. 
DEN NELL, R., 1960. Integument and exoskeleton. Pages 449-469 in Waterman, T. H., Ed., 

The Physiology of Crustacea, Vol. I. Academic Press, Inc. New York. 

FI.ORKIN, M., 1960. Blood chemistry. Pages 141-154 in Watte rman, T. H., Ed., The Physiol- 
ogy of Crustacea, Vol. I. Academic Press, Inc. New York. 
KIRBY, H., 1950. Materials and Methods in the Study of Protozoa. University of California 

Press, Berkeley and Los Angeles, 72 pp. 
MIYASHITA, Y., 1933. Studies on a fresh water foettingeriid ciliate, Hyalospira caridinac n.g., 

n.sp. Jap. J. Zool, 4 : 439-460. 
PASSANO, L. M., 1960. Molting and its control. Pages 473-536 in Waterman, T. H., Ed., 

The Physiology of Crustacea, Vol. I. Academic Press, Inc. New York. 
TRACER, W., 1957. Excystation of apostome ciliates in relation to molting of their crustacean 

hosts. Biol Bull., 112: 132-136. 



Reference : Biol. Bull, 143: 222-233. (August, 1972) 



LOSS OF LIMBS AS A STIMULUS TO ECDYSIS IN 
BRACK YURA (TRUE CRABS) 1 

1 )( )ROTHY M. SKINNER AND DALE E. GRAHAM 2 

Biology Division, Oak Ridge National Laboratory; Marine Biological Laboratory, 

Woods Hole, Massachusetts; and The University of Tennessee-Oak Ridge 

Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830 

The common means of inducing precocious molts in Crustacea is by eyestalk 
extirpation (Zeleny, 1905), which removes the X-organ-sinus gland complex con- 
taining the postulated molt inhibitory hormone (MIH; Passano, 1960) and pre- 
sumably allows the action of ecdysterone to initiate molt preparations. This pro- 
cedure, though effective in stimulating molting in many species, frequently kills 
the animals either at the time of surgery or at the time of ecdysis itself. Such is the 
case with one of the experimental animals used in our laboratory, the land crab, 
Gecarcinus lateral is. Moreover, the injection of ecdysterone into unoperated 
animals frequently leads to the death of the animals even in species where it is an 
effective stimulus to molting (Krishnakumaran and Schneiderman, 1968; Williams, 
1968; Skinner and Graham, 1970). Previously we reported that in Gecarcinus 
the loss of a large number of limbs, either pereiopods (walking legs) or chelipeds 
(claws), triggered precocious but apparently physiologically normal molts with 
high survival, whereas injections of ecdysterone were ineffective (Skinner and 
Graham, 1970). 

It is known that regeneration of lost limbs occurs in the premolt period of the 
land crab (Bliss, 1956; Hodge, 1956a, 1956b ; Skinner, 1958, 1962; Skinner and 
Graham, 1970) and many other Crustacea (Emmel, 1910; Bliss, 1960; Needham, 
1965; Hay, 1966; Goss, 1969). Furthermore, the loss of numerous limbs triggers 
precocious molts in certain insects (Cameron, 1927) and the land crab (Bliss, 
1956; Skinner and Graham, 1970). On the other hand, in some cases such as the 
cockroach, Blaiclla, loss of limbs before a certain critical time can as much as 
double the interval before the next ecdysis (O'Farrell, Stock and Morgan, 1956). 

This paper represents a study of the interrelationships between molting and 
limb regeneration in the Brachyura, or true crabs. It includes observations on: 
(1) the effect of limb loss on the duration of the molt cycle of several species of 
marine Crustacea, with data on precocious molts caused by eyestalk removal in- 
cluded for comparison; (2) the minimal number of legs which must be removed 
to cause molting; (3) the effect of the total number of legs removed on the size of 
the subsequent limbs regenerated (regenerates) ; (4) the effect of lack of privacy 
and of light regimen on the duration of the molt cycle (Bliss and Boyer, 1964) ; 

1 Research supported by the U. S. Atomic Energy Commission under contract with Union 
Carbide Corporation. 

2 Present address: Department of Biology, California Institute of Technology, Pasadena, 
California. 

222 



ECDYSES STIMULATED BY LIMB LOSS 

(5) the interaction between loss of regenerating limb buds in the early premolt 
period, duration of the premolt period, and re-regeneration of the lost regenerates. 
Experiments in (2), (3), (4) and (5) used the land crab, Gecarcinus lateralis, 
as the experimental animal. 

MATERIALS AND METHODS 

Specimens of the land crab, Gecarc'vnus lateralis, were maintained in the usual 
manner (Skinner, 1962). Specimens of two portunid crabs, (the blue crab, Cal- 
linectes sapidus, and the green crab, Carcinus maenas) the fiddler crabs, Uca 
piiynax and U. pugilator, and the spider crab, Libinia cmarginata, were kept in 
sea tables with running water at the Marine Biological Laboratories, Woods Hole, 
Massachusetts. They were fed mussels. 

Animals w r ere caused to autotomize limbs (Wood and Wood, 1932) by cutting 
at the merus. Eyestalks were removed by cutting at the articulating membrane. 
To increase survival, one eye was removed on day 1, the other on day 2 or later. 

A single leg was removed from control or eyestalkless specimens to permit us 
to detect an approaching molt by the progress of limb regeneration (Bliss, 1956; 
Skinner, 1962, 1965). Experiments on Gecarcinus were performed throughout 
the year; experiments on the marine crabs were performed in mid-June through 
August. 

The size of limbs was determined by measuring the external dimensions of 
the various segments as well as by weighing the limbs. Postmolt limb weights 
were determined one month after ecdysis, since the limb immediately following 
ecdysis contains only some 17% of the final intermolt quantity of tissue isolable 
one month later (Skinner, 1966a) after a period of rapid protein synthesis has 
occurred (Skinner, 1966b). 

RESULTS 
Response of marine Crustacea to limb loss 

Libinia: Neither controls (20), eyestalkless (8) nor animals with 6 or 8 legs 
missing (L. A. -- legs autotomized animals; 20, total) molted or showed any sign 
of an approaching molt. Scabs remained on the autotomy plane throughout the 
experiment, which was terminated after 10 weeks (Table I). 

Carcinus: Four animals missing 6 limbs regenerated legs and molted success- 
fully ; tw r o were killed by other animals. No other animals (i.e., eyestalkless or 
controls) showed any indications of an approaching molt. Eyestalkless specimens 
did show copulatory behavior typical of premolt animals, with the males grasping 
the females beneath them. 

Callinectes: Both eyestalkless animals and those missing 6 limbs prepared for 
ecdysis with attendant limb regeneration. Of 10 eyestalkless specimens, all sur- 
vived for several weeks after eyestalk removal, 3 survived until the late premolt 
period but only one until ecdysis (42 days after eyestalk loss). That animal died 
after having split the epimeral suture in the branchiostegite region and initiating 
emergence from the old exoskeleton. No appendages were freed at the time of 
death. The mucilaginous layer (molting fluid) was apparent. In the land crab. 



224 



DOROTHY M. SKINNER AND DALE E. GRAHAM 



TABLE I 
Effect of aulotomy of legs or eyestalk removal on the interval to ecdysis of marine crabs 



Animal 


Treatment 


Effect 


Days to ecdysis 


Libinia 
(Spider crab) 


E/S* (8)f 
6 to 8 L. A.J 









Ca rein us 
(Green crab) 


E/S (10) 
6 or 8 L. A. (6) 



+ 


48 5 (S.D.) 


Uca pugnax 
(Fiddler crab) 


E/S (24) 
6 or 8 L. A. (24) 


+ 
+ 


22 3 
25 2 


Callinectes 
(Edible blue crab) 


E/S (10) 
2 C. A. 
4 L. A. (5) 


+ 
+ 


42 
51, 51, 61, 64, 70 



* E/S == Eyestalks removed. 

t ( ) = : Number of animals in sample. 

J I,. A. == Legs autotomized. 

Gecarcinus, eyestalkless animals commonly die slightly later, when they have 
freed themselves almost completely from the old exoskeleton. 

The interval to ecdysis was longer in animals triggered to undergo precocious 
molts by limb loss than in those stimulated by eyestalk extirpation which removes 
the MIH (Table I). However, the viability of the L. A. animals was considerably 
greater. In addition to 5 animals that molted, two other groups of 6 and 10 L. A. 
animals had responded, as witnessed by extensive limb regeneration. Unfortu- 
nately, that experiment had to be terminated before the animals underwent ecdyses. 

Uca: Both U. pugnax and V . pugilator responded to both treatments. As in 
the case of Callinectes, there was a more rapid response to eyestalk removal, with 
ecdysis occurring within 20 to 25 days. The loss of 6 or 8 walking legs also stimu- 
lated premolt preparations, including limb regeneration. Ecdysis occurred approxi- 
mately 25 days after limb loss. The mortality of both groups of experimental 
animals was high, with only one eyestalkless animal surviving ecdysis. Others also 
report high mortality of eyestalkless Uca (Fingerman and Yamamoto, 1967). 
However, the positive response of the animals to limb loss was clear (See Fig. 1, 
which shows photographs of Uca and other crabs stimulated to molt by limb re- 
moval.) As with Gecarcinus, removal of one appendage of the marine crustaceans 
could be useful as an early indication of an approaching molt (Bliss. 1956; Skin- 
ner, 1962). 

Effect of eyestalk removal as compared to limb loss on precocious molts 

\Ye and others (Passano, 1960; Skinner, 1968) have for years used eyestalk 
extirpation as a means of propelling various species of Crustacea into precocious 
molts. The lethargic postoperative behavior of the animals, as well as their de- 
mise at the time of the subsequent ecdysis, indicated that although their macro- 
molecular metabolism appeared indistinguishable from that of normal premolt 
animals (Skinner, 1965, 1966a, 1966b), nonetheless their premolt period was ab- 



ECDYSES STIMULATED BY LIMB LOSS 



225 




FIGURE 1. Specimens of crabs with regenerating limb buds, (a, b ) Gecarcinus latcralis. 
(c) Callinectes sapidits and (d) L'cu piti/iut.r. Eiglit, 4, 6, and 6 walking legs were removed 
from animals (a, b ) 45, (c) 3o, and (d) 20 days previous to the time of photography. 



normal. We have performed a series of experiments on (.iecarcnnis to compare 
the relative effectiveness of eyestalk extirpation and limh loss in inducing pre- 
cocious molts. 

We find that eyestalk removal leads to the most rapid molting preparations 
(Table I) but is accompanied with 100^ mortality at the time of ecdyses (Fig. 2). 
Molts induced by limb loss, though somewhat delayed in time, almost always lead 
to healthy, normal animals ( Fig. 2). Even those specimens deprived of 8 walk- 
ing legs, though clumsy in their movements, molt successfully almost 100^ of the 
time. Indeed, in most experiments, we observe no deaths following such treatment. 

Minimum number oj limb autotomies required to stimulate precocious moltiruj 
preparations 

These and the remaining experiments in this paper were performed with 
Gecarcinus. 

Loss of from 1 to 4 limbs had no effect on the duration of the molt cycle 
(Table II). Animals which have lost 5 limbs, on the other hand, respond except 
when kept in community tanks. Loss of from 6 to 8 limbs, including various com- 
binations of chelipeds and pereiopods, is almost lOO^r effective. Since in 
Gecarcinus the chelipeds comprise as much as 35 c /c (21 to 35 r /r ; Table III) of 
the total body weight, whereas the walking legs contribute 11 to \7 c /c (Table III), 



226 



DOROTHY M. SKINNER AND DALE E. GRAHAM 



TABLE II 

Effect of autotoniy of legs on interval to ecdysis in Gecarcinus. Abbreviations arc: 

L. A. = walking leg autotomized; C. A. = cheliped autotomized; (a) == In 

jar, ca. 10 win light week; (b) == In jar, ca. 10 hr light /week; 

(c) == Community tank (6-8 animals); JO hr light 'week 







Treatment 


Animals 

(No.) 


Mean time to ecdysis 
(Days S.D.) 


1 1.. 


A. 


(a) 


14 


>2()() 






(c) >tork Mipplv 


>30Q 


>200 


2 C. 


A. 


(a) 


6 


>!()() 


4 L. 


A. 


(a) 


6 


>\65 


5 I.. 


A. 


(a) 


7 


86 17 






(1.) 


8 


86 8 






(c) 


8 


>\3\ 


2 C 


. , 


4 L. A. (a) 


8 


77 25 


6 L. 


A. 


(a) 


8 


8Q 18 


8 L. 


A. 


(a) 


8 


70 12 






(b) 


7 


81 17 



loss of two chelipeds should have been effective in stimulating molting if the stimulus 
were loss of tissues mass. However, such was not the case. 

The duration of the premolt period is dependent on the size of animals, being 



lOO-i 



80- 



co 

UJ 
CO 

>- 

Q 
CJ 

LJ 



60- 



40- 



20- 








20 40 60 80 

TIME(days) 



100 120 



FIGURE 2. Effect of eyestalk removal or autotomy of limbs on the duration of the inter- 
molt period of Gecarciui/s. Eyestalks were removed on successive days from 7 animals . 
all of which died at ecdysis; 8 walking legs were autotomized from 15 animals D D. The 
animals were housed in individual bottles and observed until ecdysis occurred. 



ECDYSES STIMULATED BY LIMB LOSS 



227 



TABLE III 

Per cent body weight comprised by claws or walking legs of Gecarcinus lateralis. 
Animal 3 -was female; all others were males 







% Body weight 




Total weight 






(Grams) 










2 Chelipeds 


8 Pereiopods 


Animal 1 


53.0 


77 


17 


j 


60.4 


22 


15 


3 


63 


21 


11 


4 


64.5 


27 


14 


5 


67 


31 


16 


6 


96 


35 


13 


7 


9Q 


32 


17 



longer for large animals than for small. For example, animals of carapace width 
4.2 to 4.7 cm underwent ecclyses 33 to 54 days after limb autotomy, whereas 
animals of carapace width 5.2 to 5.3 cm took 50 to 82 days. Therefore, in any 
one experiment, animals of similar size were used. 

Environmental effects on the length of the molting cycle 

When crabs lacking either 6 or 8 walking legs are maintained in bottles, which 
are stored in relatively light-tight cabinets, they receive approximately 10 min of 
light per week. Such animals molt sooner than those maintained in community 
tanks that receive 10 to 12 hours of light per week during the routine cleaning 
and feeding operations ( 'Fable II ). This observation confirms a previous report of 
Bliss and Boyer ( l c '(>4). The following experiment demonstrated that the effect 
was due to privacy rather than the amount of exposure to darkness. A series 
of animals was maintained in bottles kept outside the cabinets usually used for 
storage of experimental animals. Thus these animals received the same exposure to 
light as did the animals kept in groups of 5 or 6 in community tanks. These 
animals molted as soon as did animals kept in bottles in the dark (Table II). 

It should be noted that the marine crabs studied were maintained in com- 
munity tanks. Since limb removal stimulated precocious molts in all species of 
marine animals (Callinectes, Cancer, Uca) except Libinia, it may be that Libinia 
is more sensitive to the presence of other animals than the other marine crabs. 



Effect of total number of liinl's removed on the she of the regenerate 

Regenerated limbs are somewhat smaller than their non-regenerated counter- 
parts. Ouantitation of the size difference leads to the following observations : 
(Table IV) Compared to those of bilaterally symmetrical partners, the weights of 
non-regenerated limbs taken from 29 animals varied by no more than 4%, whereas 
the weights of regenerated limbs were smaller by approximately ^ when from 1 
to 6 limbs were removed. Since there are no normal, bilaterally symmetrical 
limbs left on crabs that have been induced to regenerate 8 walking legs, the weight 



228 



DOROTHY M. SKIXNER AND DALE E. GRAHAM 



TABLE IV 



Effect of the removal of one to eight limbs on the size oj regenerated limb. Regenerative load refers 

to the number of limbs regenerated during a molting period. One or more months after 

ecdysis, limbs were removed and weighed. Regenerated legs were compared to 

bilaterally symmetrical non-regenerated legs. To test individual variability, 

crabs were induced to aiitotomize a pair of normal limbs. The data on 

X L. A. animals were obtained by comparing the weights of legs 

of normal animals of the same size (see text) 



Regenerative load 
(walking legs) 


Percentage change in 
weight between any 
two walking legs 


Sample 
size 


0; (individual variability) 


4.4 


2Q 


1 


-34.5 


6 


6 


-34.7 


16 


8 


-48.4 


9 



of such legs \vas compared to the weight of non-regenerated (normal) legs of a 
series of crabs of the same carapace width, a fixed parameter useful for comparing 
animals of different stages of the molt cycle. Loss of X walking legs leads to a 
striking decrease in the mass of tissue contained in each limb (Table IV). Rough 
calculations of the mass of tissue regenerated indicated that an animal (of the sizes 
used in these experiments ) was capable of synthesizing approximately 4 grams of 
tissue which was distributed among the regenerated appendages. Thus, if (> limbs 
were regenerated, each weighed y\ less than non-regenerated partners whereas if X 
were regenerated, each weighed ^ that of its non-regenerated control. 



Effect oj removal oj partially regenerated limbs on the time oj ecdysis 

In another series of experiments (Skinner and Beattie, 1971), we have been 
studying the metabolic and biosynthetic properties of regenerating limbs. For those 
experiments, we cause the animals to aiitotomize regenerating limbs at various 
stages in their development. In one of our early experiments, limb buds of the 
same external dimensions and appearance were removed from 3 crabs. Two of 
the crabs molted within 10 days (see Animals 6 and 7, Fig. 3), without regenerat- 
ing the limbs removed. The third crab, which molted 35 days after the removal 
of its limb buds, had regenerated those removed (Animal 19, Fig. 3). Apparently, 
there was a delay in the time of ecdysis sufficient to permit reformation of the 
limbs. 

Since the length of time from one ecdysis to the next is not clearly defined in 
the land crab as it is for ecdyses specific to particular insect larval instars, it is 
not possible to determine the magnitude of the retardation in time of ecdysis with 
certainty. However, in observations on 30 animals ( Fig. 3 ) , no animal which 
underwent ecdysis within 25 days after loss of partially regenerated limbs had 
replaced those removed. By contrast, all animals which molted 33 or more days 
after autotomy of limb buds had re-regenerated. Thus the shortest time required 
for re-regeneration was 33 days after autotomy of partially regenerated limb buds. 
This corresponds favorably with the time required from eyestalk removal to ecdysis. 



ECDYSES STIMULATED BY LIMB LOSS 



229 



DAYS TO AUTOTOMY OF 
LIMB BUDS 



DAYS TO ECDYSIS 



60 



ANIMAL 
NUMBER 

I 



5 



LIMB BUDS 

NOT REREGENERATED 
REREGENERATED 



10 



15 



20 



25 



30 




FiGL'KE 3. Effect of removal of regenerating limb buds on the duration of the premolt 
period of Gccarcinns. Molting preparations were initiated in 30 animals by limb removal and 
at some later date (Day in this figure) regenerating limb buds were autotomized (L. B. A.). 
Animals were observed to the next ecdysis. The total length of the horizontal bar represents 
the duration of the complete premolt period. At the time of the next ecdysis it was noted 
whether the limbs had been re-regenerated. For purposes of graphical presentation here the 
animals are ordered into 2 groups: (1) numbers 1 through 16 are those that did not re- 
regenerate; (2} 17 through 30 are those that did. Within each group, the animals are listed 
from the longest to the shortest interval between initiation of molt preparations and limb bud 
autotomy. Note that the duration of the premolt period in animals that did not re-regenerate 
(Group 1) varied from 38 to 70 days, while in those that did re-regenerate ( Group 2) the 
premolt period varied from 60 to 120 days. The cross hatched bar indicates the critical 
period (see text). 



230 DOROTHY M. SKINNER AND DALE E. GRAHAM 

with attendant limb regeneration (Fig. 2). which was 27 days in animals of similar 
size. 

DISCUSSION 

We have now demonstrated that the loss of a large number of limbs causes 
precocious molts not only in the land crab but also in three species of marine 
crabs. Two of these also prepare for ecdysis soon after eyestalk removal (Cal- 
lincctcs and Uca ) but frequently die at the time of or shortly after ecdysis. The 
third (Carcinits} does not prepare for molting after eyestalk removal if greater than 
a certain size (approximately 2 cm carapace width, Bauchau, 1961; these data). 
Our experiments indicate that in large specimens of the green crab, limb loss is a 
more effective stimulus to ecdysis than is eyestalk removal. 

Our experiments on the marine crabs, though preliminary, show that precocious 
molts triggered by loss of a critical number of limbs lead to apparently normal, 
healthy ecdyses, which the animals survive with as high viability as from normal 
ecdyses. Thus, this appears to be the method of choice for inducing precocious 
molts. 

We do not suggest that these experiments demonstrate the presence of a molt- 
inhibitory factor in the limbs of Crustacea. A more likely hypothesis is that the 
stimulus to precocious molts is due to the severing of a critical number of nerves. 

It would be of interest to attempt to stimulate precocious molts by severing 
the nerves to the walking legs of crabs. We expect that these experiments would 
prove to be technically difficult because of the rapidity with which the animals, at 
least Gccarcinus, autotomize limbs when confronted with noxious stimuli, such 
as anesthetization by exposure to the cold (4 C ) or the injection of materials 
into the base of the appendage. 

Cameron (1927) found that the interval between molts in .-mother arthropod, the 
centipede Scutigera jorca, was shortened by one half the normal time when all its 
legs were removed. Loss of as many as 26 of the 30 legs was ineffective in 
shortening the duration of the molting cycle. Unlike Scntnjcra, loss of all limbs 
apparently inhibits molting preparations in Gccarcinus. Two of our specimens of 
Gccarcinus lost all of their limbs in combat with other crabs. Both had normal eye 
reflexes and bore no evidence of other bodily injury. Yet, there were no signs 
of regeneration after 3 months, although other animals from the same shipment 
missing 6 or 8 walking legs had long since regenerated their lost limbs and under- 
gone ecdyses. Since specimens of Gccarcinus respond within 3 weeks by basal 
growth of autotomized appendages, and since the limbless animals were hand-fed, 
it is unlikely that starvation was the explanation for their lack of response, especially 
since Cameron (1927) found that starved specimens of Scutigera survived one 
ecdysis and lived well into the next intermolt period (approximately 60 days). 

A phenomenon similar to the critical period for re-regeneration of limbs in 
Gccarcinus (Fig. 3) was reported in the insect. Blatclla (O'Farrell and Stock, 
1953). Under conditions when the duration of the first instar was 12 days (25 
C), loss of one limb between days 1 and 4 increased the period until the next 
ecdysis by 3 days. The total duration of the instar was then 15 instead of 12 days. 
At higher temperatures (30 C), the intermolt duration was increased from 5 to 



ECDYSES STIMULATED BY LIMB LOSS 231 

6 days by the loss of one limb. A limb lost after a cretain critical period was not 
regenerated by the time of the next ecdysis. Rather, it remained as a papilla, to 
be regenerated during the next premolt period (O'Farrell and Stock, 1953). 

The crustacean premolt period may be subdivided into 5 parts, designated stages 
D,, through D 4 (Drach, 1939; Skinner, 1962, 1966a). A substantial fraction, if 
not all, of limb regeneration occurs in D,, (Skinner, 1962). It has long been 
known that there is a critical time in the arthropod premolt period (O'Farrell ct al., 
1956; problably D in crustaceans; Skinner, 1962, 1966a) before which limbs must 
be removed if they are to be regenerated by the first ecdysis following limb loss. 
The ability of an animal to prolong D,,, in which it synthesizes another set of limbs 
and concomitantly delays the next series of premolt events (such as apolysis, the 
separation of the epidermis from the old exoskeleton, Jenkin. 19/0; resorption of 
the old exoskeleton and synthesis of a new exoskeleton, Skinner. 1962) points up the 
complexity of the controls over various premolt phenomena. Our observations 
indicate that even if a single "on" switch is thrown to initiate the premolt period, 
the ordered series of events leading to a normal ecdysis do not proceed on an 
invariant time schedule. The whole premolt process appears to be self-monitored in 
such a way that subsequent events cannot proceed until early events reach a 
critical stage of completion and the duration of the critical period can be greatly 
extended. In fact, we think that the animals that did not re-regenerate were already 
in stage D t (and that the epidermis had already separated from the old exoskeleton 
when their regenerating limb buds were autotomized ). Clearly, there is no turn- 
ing back. The interacting controls are such that the animals did not even initiate 
the re-regeneration process. 

We wish to thank W. G. Beattie and E. Ang for their help with some of these 
experiments. Some experiments were performed by the senior author in conjunc- 
tion with the Experimental Invertebrate Course at the Marine Biological Labora- 
tory, Woods Hole, Massachusetts. The help of the collecting staff and students in the 
course was greatly appreciated, as were the photographs of Callincctcs and Uca, 
taken by Mr. P. J. Oldham of the Systematics-Ecology Program. I). E. Graham 
was an Oak Ridge Graduate Fellow under appointment from the Oak Ridge Asso- 
ciated Universities. 

SUMMARY 

1. Loss of 6 to 8 pereiopods or chelipeds triggers precocious molts in a num- 
ber of marine crabs including the green crab, Carciniis inacnas, the blue crab, 
Ca/lincctcs sapidits, and the fiddler crabs, l j ca piKjna.v and U. pugilator, but not 
in the spider crab, Libinia cinari/inata. Mortality rates are negligible compared to 
those of animals induced to molt by eyestalk removal. 

2. Precocious molts can be elicited in the land crab, Gccarciniis lateralis, by the 
loss of 5 to 8 appendages (pereiopods and chelipeds) but the loss of all 10 append- 
ages inhibits molting. Loss of a cheliped which in Gccarciniis may have a mass 
ten times greater than a pereiopod is no more effective than loss of a walking leg. 

3. The size of the regenerates formed in Gccarciniis is reduced by one-third 
from normal size when from 1 to 6 pereiopods are lost, and by one-half when 8 
limbs are regenerated. 



232 DOROTHY M. SKINNER AND DALE E. GRAHAM 

4. When one or more partially regenerated limbs is removed before a certain 
critical time in the premolt period (Stage D,,?) the animal re-regenerates replace- 
ment appendage ( s ). This results in significant lengthening of the interval before 
ecdysis occurs. 

Note added in proof: Dr. (ieorge D. Bittner (personal communication) finds 
that in the crayfish, Procanihonis clarkii. cutting of the nerves to 6 pereiopods is 
effective in shortening the interval between two ecdyses. In this species nerves 
to the pereiopods can be cut without the induction of autotomy. 

LITERATURE CITED 

BAUCHAU, A. (',., 1%1. Regeneration des Pereiopods et croissance chez les Crustaces De- 

capodes Brachyoures. .-Inn. Sue. Roy Zool. Bel<i.. 91 : 57-84. 
BLISS, D. E., 1956. Neurosecretion and the control of growth in a decapod crustacean. Pages 

56-75 in l\. (i. Wingstrancl, Ed., Bcrtil Hanstram: Zoological Papers in Honour of 

his Si.vty-fiith Birthday, November 20, 1956. Zoological Institute, Lund, Sweden. 
BLISS, D. E., 1%0. Autotomy and regeneration. Pages 561-589 in T. H. Waterman, Ed., 

The Physioldi/y of Crustacea. I. Metabolism and Gro-wth. Academic Press, NY\\ 

York and London. 
BLISS, D. E., AND J. R. BOYER, 1%4. Environmental regulation of growth in the decapod 

crustacean Geeareinns Intertills, Gen. and Comp. Endocrinol.,4'. 15-41. 
CAMERON, J. A., 1927. Regeneration in Sentii/era forceps. J. E.rp. Zool.. 46: 169-179. 
DRACH, P., 1939. Mue et cycle d'intermue chez les Crustaces Decapodes. .Inn. Insf. Occanm/i. 

Monaco, 19: 103-391. 
EMMEL, V. E., 1910. A study of the differentiation of tissues in the regenerating crustacean 

limb, .liner. J. Anat.,10: 109-159. 
FINGERMAN, M., AND Y. YAMAMoTO, 1967. Daily rhythm of melanophoric pigment migration 

in eyestalkless crabs, Uca pui/ilator (Bosc). (.'rustaceana. 12: 303-319. 
Goss, R., 1969. Principles of Rei/eneration. Academic Press, New York and London, 287 

pages. 

HAY, E. D., 1966. Regeneration. Holt, Rinehard and Winston, New York, 148 pages. 
HODGE, M. H., 1956a. Autotomy and regeneration in Gccnrciints lateralis. Anat. Ree., 125: 

633. 
HODGE, M. H., 1956b. Variations on the normal pattern of limb regeneration in Geeareinns 

I,, I. -rails. Anat. Ree.. 125: 635-636. 
JEXKIN, P. M., 1970. Part II: Animal Ifonixnies. Control of Growth and Metamorphosis. 

Pergamon Press, New York. 

KRISHNAKUMARAN, A., AND H. SCHNEIDERMAN, 1968. Chemical control of moulting in arthro- 
pods. Nature. 220: 601-603. 
NEEDHAM, A. E., 1965. Regeneration in the arthropoda and its endocrine control. Pages 283- 

323 in V. Kiortis and H. A. L. Trampusch, Eds., Regeneration in Animals and Re- 
lated I'roblenis. North-Holland Publishing Co., Amsterdam, Netherlands. 
O'FARRELL, A. F., AND A. STOCK, 1953. Regeneration and the moulting cycle in Blatella 

i/ennanica L. I. Single regeneration initiated during the first instar. Austr. J. Biol. 

Sci., 6 : 485-500. 
O'FARRELL, A. F., \. Sin, K .\XD J. \IoRGAN, 1956. Regeneration and the moulting cycle in 

Hlntelln fii-nnaniea L. IV. Single and repeated regeneration and metamorphosis. Austr. 

J. Biol. Sci., 9: 406-422. 
PASSANO, L. M., I960. Molting and its control. Pages 473-536 in T. H. Waterman, Ed., The 

Physiology of Crustacea. I. Metabolism and Growtli. Academic Press, New York 

and London. 
SKINNER, D. M., 1958. Structure and metabolism of a crustacean tissue during a molt cycle. 

Ph.D. thesis. Harvard l'ni;-ersity. Ill pages. 
SKINNER, D. M., 1962. The structure and metabolism of a crustacean integumentary tissue 

during a molt cycle. Biol. Bull.. 123 : 635-647. 



ECDYSES STIMULATED BY LIMB LOSS 233 

SKINNER, D. M., 1965. Amino acid incorporation into protein during the molt cycle of the 

land crab, Gecarcinus lateralis. /. Exp. Zool., 160 : 225-233. 
SKINNER, D. M., 1966a. Macromolecular changes associated with the growth of crustacean 

tissues. Amer. Zool., 6 : 235-242. 
SKINNER, D. M., 1966b. Breakdown and reformation of somatic muscle during the molt cycle 

of the land crab, Gecarcinus lateralis. J. Exp. Zool., 163 : 115-124. 
SKINNER, D. M., 1968. Isolation and characterization of ribosomal ribonucleic acid from the 

crustacean, Gecarcinus lateralis. J. Exp. Zool., 169 : 347-355. 
SKINNER, D. M., AND W. G. BEATTIE, 1971. Synthesis of satellite DNA by crustacean organs 

maintained in vitro. Cell Biol. Abstracts, 1971 : 278. 

SKINNER, D. M., AND D. E. GRAHAM, 1970. Molting in land crabs : stimulation by leg re- 
moval. Science, 169 : 383-385. 
WILLIAMS, C. M., 1968. Ecdysone and ecydsone-analogues : Their assay and action on dia- 

pausing pupae of the Cynthia silkworm. Biol. Bull., 134 : 344-355. 
WOOD, F. D., AND H. E. WOOD, 1932. Autotomy in decapod Crustacea. /. Exp. Zool., 62: 

1-55. 
ZELENY, C., 1905. Compensatory regulation. /. Exp. Zool., 2 : 1-100. 



Reference: Biol Bull, 143: 234-246. (August, 1972) 



WATER-EXCHANGE IN THE CRAB HEMIGRAPSUS NUDUS 

MEASURED BY USE OF DEUTERIUM AND 

TRITIUM OXIDES AS TRACERS 

RALPH I. SMITH * AND PAUL P. RUDY 

' Oregon Institute of Marine Biology, University of Oregon, 
Charleston, Oregon 97420 

The water-permeability of several decapod crustaceans has been measured by 
Rudy (1967) using tritiated water (THO) as tracer. Among the animals studied 
was the Atlantic green crab, Carcinus macnas, a good osmotic regulator that has 
been the subject of many physiological investigations. Smith (1970) restudied 
Carcinus iitacnas, using deuterium oxide (DHO) as tracer, and made estimates of 
the water exchange rate and net water influx. Water-exchange rate was found 
to decrease with the salinity of the medium, as had previously been demonstrated 
for the very euryhaline crab Rhithropanopeus harrisi (Smith, 1967). Smith's 
results on Carcinus, based on DHO, seemed to indicate a markedly higher water- 
exchange rate than did the results of Rudy (1967), based on THO as tracer. Even 
after making reasonable corrections for differences in temperature and in the 
size of experimental animals, there remained a discrepancy in the hourly water- 
exchange fraction, with the values based on DHO about 20% higher than those 
based on THO. In this connection it was considered possibly significant that 
water-exchange values for the prawn Palaemonetes varians by Rudy (1967) using 
THO averaged only half the value obtained by Parry (1955) using DHO. It 
was further noted that, in unpublished studies, Smith had obtained a comparable 
20 % discrepancy between the water fluxes calculated from studies on Rhithro- 
panopeus harrisi, using DHO and THO as tracers in separate experiments. Taken 
together, the above three sets of experiments suggest an isotope effect, but no 
quantitative value should be assigned because of the chain of assumptions involved 
in the estimates. 

In considering the plausibility of an isotope effect between DHO and THO, it 
is improbable that it could be attributed to differences in diffusion rate among 
isotopic molecules of water as such. The molecular dimensions of D 2 O and H 2 O 
are almost identical (Kavanau, 1964), suggesting similar molecular diffusion 
rates. Deuterium and tritium oxides in dilute solution are almost entirely in the 
form of DHO and THO, with molecular weights of 19 and 20, not greatly different 
from the weight of 18 for H 2 O. Wang, Robinson and Edelman (1953) found that 
the diffusion coefficients of DHO and THO in H 2 O did not differ significantly, 
but that the diffusion coefficient of H 2 O 18 w^as about 14% higher than the values 
for DHO and THO. Chinard and Enns (1954) found no difference in the rates 

1 Address of senior author : Department of Zoology, University of California, Berkeley, 
California Q4720. 

234 



WATER-EXCHANGE IN CRAB HEMIGRAPSUS 235 

of passage of DHO and THO across capillary walls in the dog, and Enns and 
Chinard (1956) likewise found no significant difference in the passage of THO 
and H 2 O 18 in this experimental situation. Nevertheless, Johnson and Babb find 
it necessary to observe (1956, page 442), "It is noted that serious disagreement 
exists among various investigations of the self-diffusion coefficients of water" (in- 
volving the use of THO, DHO, and H 2 O 18 as tracers). Paganelli and Solomon 
(1957), noting the results of Wang et a!., (1953), increased their estimates of H 2 O 
fluxes into the red cell, calculated on the basis of THO fluxes, by 14% to com- 
pensate for the reported difference in diffusion coefficients. Thus the matter of 
differences in the diffusion rates of THO, DHO, and H 2 O cannot be regarded 
as settled. 

A second possibility for explaining an isotope effect between the uptakes of 
DHO and THO is that these isotopes might themselves interact with biological 
membranes in such a way as to lower water-permeability. Kavanau (1964) points 
out that the structural order and degree of hydrogen bonding are greater in liquid 
D 2 O than in ordinary water, and it may be inferred that the bonding of T 2 O is 
even stronger. But the experiments upon crustaceans cited in the present paper 
involved the use of D 2 O at a concentration of 5%, while ToO was used at a con- 
centration of only 0.1%, hence it is unlikely that a measurably lower uptake of 
THO could be attributed to effects of the tritium isotope upon the membranes 
themselves. Although it may be possible that different species of crustaceans can 
distinguish between the chemically identical DHO and THO and show different 
DHO/THO permeability ratios, no evidence for such is known to the authors. 
It would have been most desirable to restudy Carcmus, but this Atlantic species 
was not available. 

Tf an isotope effect of the magnitude reported is not likely to have resulted 
either from diffusion rate differences among isotopic water molecules, or as a 
consequence of alteration of the permeability of the membranes involved, attention 
should be directed to possible reactions involving the atoms or ions of the hydrogen 
isotopes themselves. Protium, deuterium, and tritium differ in mass as 1:2:3. The 
mobility of the ions H f , D+, rind T f in water is regarded as not simply the diffusion 
of these ions as hydrated particles among water molecules, but as a process of proton 
transfer (cf. Pimentel and McCllelan, 1960, page 254). In this process the isotopic 
ions of D 2 O may exchange rapidly between water molecules with the formation of 
D f , H + , OH~, H 3 O f , and H 2 DO + ions. The transfer of hydrogen and its isotopes 
from molecule to molecule may be expected to be mass-dependent and, to the ex- 
tent that it is involved in the penetration of deuterium and tritium tracers into 
animals, might account for an observed slower uptake of THO as compared to DHO. 
In consequence, the DHO tracer would indicate a higher rate of water exchange 
than is indicated by THO, and the actual exchange of H 2 O might be higher than is 
indicated by the use of either of the heavier isotopic tracers. Until such problems are 
clarified and a better quantitation made of any possible H 2 O/DHO/THO isotope 
effect, water-permeability values obtained by use of different isotopes of water can- 
not be directly compared and the possibility must be borne in mind that neither 
DHO nor THO may indicate the full value of the permeability of animal surfaces 
to H 2 O. 

In order to clarify the discrepancy between our results (Rudy 1967, Smith 



236 RALPH I. SMITH AND PAUL P. RUDY 

1970), we have carried out a double-tracer study with DHO and THO. This 
should eliminate variables of temperature and season as well as of size, sex, and 
moult stage of the animals used, and should minimize individual operational varia- 
tion. We hoped that these experiments would allow us to quantify an isotope 
effect if such existed, or to rule it out as experimental artifact, and to provide 
an empirical check on the results given by the THO and DHO methods of deter- 
mining water fluxes as we have used them. 

MATERIALS AND METHODS 

This joint study was carried out in late August and early September, 1970, at 
the Oregon Institute of Marine Biology, University of Oregon, Charleston, Oregon. 
Hemigrapsus nudus (Dana) was selected as the most readily obtainable euryhaline 
crab, not unlike Carcinus in its ecological preferences, and available in a wide size 
range. Experimental animals were maintained in running seawater (SW) at 
12-13 C, or in 8-inch fingerbowls of SW diluted with tap water and kept cool in 
running SW. The local SW, taken near the mouth of the estuary of the Coos River, 
had an osmotic concentration of 1009 milliosmoles at the start of the study, drop- 
ping with the onset of rains to 960-980 milliosmoles. The osmotic pressures of 
blood serum and experimental media were determined by use of a Hewlett-Packard 
Vapor Pressure Osmometer, model 302B, with NaCl standard of 1000 milliosmoles. 
Blood was collected in amounts of 0.7 to 1 ml with a fine-tipped glass pipette, and 
discharged into a very small test tube, which was stoppered and placed in boiling 
water for 10 seconds. The firm white clot which formed was broken up with 
a slender glass rod, and the sample centrifuged to make the clear serum available. 
The blood of T. nudus clots so strongly that attempts to carry out VPO determina- 
tions on whole blood were unsuccessful. Determinations of osmotic pressure of 
serum as a function of external salinity were carried out on randomly selected crabs 
not used for the DHO-uptake studies, since the presence of even a small per- 
centage of DHO, as a second volatile solvent, renders VPO determination impos- 
sible. 

For studies of uptake of DHO and THO, 5% by volume of D 2 O (Bio-Rad 
Laboratories, Richmond, California, 99 moles %} was added to the SW (giving 
"95% SW"), or to SW diluted with glass-distilled water (DW) so as to approxi- 
mate an osmotic concentration of 600 milliosmoles ("60% SW"). To each liter 
of the above "5% D 2 O" solutions was added 1 ml of tritiated water (THO) to 
given an activity of 1 ju,c per liter. 

Exposure of crabs to the above THO/DHO solutions was carried out by one 
of us (R. I. S.) following as closely as possible the procedure used by Smith 
(1970) on Carcinus viacnas. Tests were made in each salinity, at 10 and 20 C. 
Temperature variation was held within limits of 0.25. Exposures were for 15 
minutes, at the end of which each crab was rinsed quickly in isotope-free medium, 
wrapped and blotted in absorbent paper, and a blood sample drawn by puncture 
of the arthrodial membrane at a leg base. A few drops of this sample were imme- 
diately placed in the large end of a Pasteur pipette and distilled at 50 C, as in the 
experiments on Carcinus (Smith, 1970) and as described in detail in Welsh, Smith, 
and Kammer (1968), for determination of DHO. This, and subsequent calcula- 



WATER-EXCHANGE IN CRAB HEMIGRAPSUS 



237 



tions of the rate of DHO-uptake as a function of body weight, the determination 
of the hourly water-exchange fraction (K) on the basis of DHO-uptake, and the 
determination of Q 10 for DHO-uptake were carried out by R. I. S. The remainder 
of the blood sample was discharged into a small screw-capped jar and frozen. 
These samples were then vacuum-distilled by P. P. R. in a freeze-drying apparatus, 
as nearly as possible following the method he had used in his study of Carcinns 
(Rudy, 1967), and the THO-saturation (% specific activity) of the blood deter- 
mined with a Nuclear Chicago scintillation counter. Further treatment of these 
data was carried out by R. I. S. in order to eliminate any slight methodological 
differences. 



1300 



1200 



100 



1000 

"5 

E 

to 
O 

: 900 



o> 
to 



800 



-a 
o 

2 700 
CD 



600 



400 





sw 



400 600 700 800 900 1000 

Medium, milliosmoles 



noo 



1200 



FIGURE 1. Osmotic concentration (milliosmoles) of blood serum of Hcwignipsus 
as a function of concentration of the medium. 



238 



RALPH I. SMITH AND PAUL P. RUDY 



RESULTS 
(a.) The osmotic pressure of blood serum as a function of salinity 

One of the discrepancies between the results of Rudy (1967) and Smith (1970) 
regarding water-uptake in Carcinus lay in the fact that the authors found different 
concentrations of chloride in animals adapted to SW, and a relationship between 
chloride concentration and osmotic pressure of blood had to be assumed on the 
basis of possibly inappropriate data of earlier authors. VPO determinations of 
osmotic concentration of serum were agreed upon as a better basis for the estima- 
tion of the mole fractions of water in blood of animals adapted in 60% and 95% 
SW. The results (Fig. 1) show that the blood of Hemigrapsus nudus is iso- 
osmotic to the medium at about 980 milliosmoles (ca. 98% SW) under the con- 
ditions of the experiments, and hypertonic (ca. 865 milliosmoles) to the medium 
(ca. 600 milliosmoles) in 60% SW. From 100% to 120% SW, the blood is 
iso-osmotic with the medium. Animals in 50% SW showed signs of being over- 



100 

90 
80 
70 
60 

50- 



40- 



30 

c 25 



20- 



15 - 



- 90 
"5 80 

3 70 
"5 60 

50 
o^ 40 

30 

Q 25 

20 



10 



I I I 1 1 r 



A. 60 % Sea water 
OHO 




i i i i 



B. 95% Seawater 
DHO 




20C 



IOC 



. L 



4 5 6 7 8 9 10 



20 



30 40 



60 80 100 



Wet weight in grams 



FIGURE 2. Uptake of DHO by Hemigrapsus nudiis in a 15-minute exposure, as a func- 
tion of salinity, temperature, and body weight, expressed as per cent of the concentration of 
DHO in medium. 



WATER-EXCHANGE IN CRAB HEMIGRAPSUS 



239 



stressed, some deaths occurred, and the curve of osmotic regulation showed a notice- 
able drop below the level of the regulatory plateau. 60% SW, as used in the iso- 
tope-uptake tests, appeared to be within the physiologically acceptable regulatory 
range. 

( b. ) The uptake of DHO 

The concentration of DHO attained in the blood in a 15-minute exposure in 
60% and 95% SW was determined at 10 and 20 C. Animals in three of the 
groups numbered 34, 41, and 31, with wet weights ranging from less than one to 
more than 40 grams. Plots of uptake against weight (Fig. 2 and Table I) yielded 
curves of comparable slopes (b-1 averaging -0.0766). However, in one group, 
numbering 27 and tested in 95% SW at 20 C, the weight range was only 3.7 to 
16 grams. The slope (b-1 = - 0.2091) is quite different from the rest, and is 



TABLE I 

Part A: Uptakes of THO and DHO by Hemigrapsus nudus under four experimental conditions, with 

probabilities of differences in uptake related to type of isotope being significant 

("t" test), calculated for wet weight of 10 g. Part B: Significance 

of differences in uptake in respect to salinity 

Part A 



Experimental conditions 


n 


Isotope 


Mean uptake as 
% Sat/ 15 min 


Standard 
deviation 


Probability 


95 % SW 


20 C 


27 


THO 


29.92 


3.69 


Not significant 


DHO 


29.66 


4.16 


10 C 


31 


THO 


20.93 


3.76 


Not significant 


DHO 


21.51 


3.46 


60% SW 


20 C 


41 


THO 


26.73 


4.34 


>0.05 


DHO 


28.50 


3.90 


10 C 


34 


THO 


17.24 


2.68 


<0.02 


DHO 


18.81 


2.56 



Part B 



Salinity 


Temp. 


n 


Isotope 


Probability 


n 


Isotope 


Probability 


95% SW 


20 C 


27 


DHO 


>0.10 


27 


THO 


<0.01 


60% SW 


41 


41 


95% SW 


10 C 


31 


<().()! 


31 


0.001 


60% SW 


34 


34 



240 



RALPH I. SMITH AND PAUL P. RUDY 



considered unreliable because of the small weight range tested. Therefore, the 
mean (b-1) value of the other three groups was used in adjusting the uptake values 
of the small fourth group to 10-g weight. This irregular procedure does not, in 
fact, change the mean value at 10-g weight very greatly, raising the mean uptake 
from 28.31% to 29.66 % saturation. 

When the uptake values of DHO (as % external DHO concentration or "% 
saturation") attained in 15 minutes are adjusted for a weight of 10 grams, which 
is not far from the mean weight of the crabs used, the values for DHO uptake 
are as shown in Figure 5 and Table I. A significant reduction of uptake is shown 
in 60% SW as compared to 95% SW at 10 C, confirming the apparent reduc- 
tion of permeability to water (more propertly, reduction of water exchange) shown 
at lower salinities by the crabs Carcinus (Smith, 1970) and Rhithropanopeus 
(Smith. 1967). However, the reduction of water-exchange in 60% SW when mea- 
sured at 20 C is not statistically significant. Probably this results from the in- 

TABLE II 

Hourly exchange fractions (K) and Qio of DHO and THO uptake by 
Hemigrapsus nudus, calculated for a wet weight of 10 g 



Experimental conditions 


n 


Isotope 


Hourly exchange fraction 
"K" 


QlO 


95% SW 


20 C 


27 


DHO 


1.41 


[1.45] 


10 C 


31 


0.97 


60' 1 ;, SW 


20 C 


41 


1.34 


1.61 


10 C 


31 


0.83 


95% SW 


20 C 


27 


THO 


1.42 


[1.51] 


10 C 


31 


0.94 


60% SW 


20 C 


41 


1.24 


1.63 


10 C 


31 


0.76 



adequacy of the sample tested at 20 C in 95% SW. Mean hourly water exchange 
fractions "K," given by the equation K" (2.3/t) Log 10 (100/100-% Sat), cal- 
culated from the mean DHO uptakes adjusted for a wet weight of 10 g are given 
in Table II, as are Q 10 values for DHO uptake estimated by the ratios of "K" at 
20 C to "K at 10 C. Since the sample tested at 20 C in 95% SW is open 
to question, the Q 10 value of 1.61 for DHO uptake in 60% SW is considered the 
more reliable of the two. 

(c.) The uptake of THO 

The concentration of THO attained in the blood in 15-minute exposures in 
60% and 95% SW was the result of the same exposures as for DHO. Plots of 



WATER-EXCHANGE IN CRAB HEMIGRAPSUS 



241 



uptake against weights (Fig. 3 and Table I) yielded curves with (b-1) values much 
like those for DHO. One high value ( 0.1486) is regarded as unreliable because 
of the small weight range of the crabs used in this group (95% SW at 20 C). 
The average of the remaining three groups was - 0.0705, and this was used in 
correcting uptake in the deviant group to that of 10-g animals. 



100 

90 
80 
70 
60 

50 
40 



30- 



25 

E 20 
in 



15- 



_ 100 

o 90 

~ 80 

E 70 

5 60 
o 

00 50 



o- 

O 30 

20 
15 

10 



A. 60 % Seawater 
THO 




20C 



r-r--ioc 



B. 95 % Seawater 
THO 




IOC 



3 4 5 6 7 8 9 10 20 30 40 

Wet weight in grams 



60 80 100 



FIGURE 3. Uptake of THO by H. nudiis in a 15-minute exposure, as a 
function of salinity, temperature, and body weight. 



When the uptake ("% saturation") values attained in 15 minutes are adjusted 
for a weight of 10 g, the values for THO uptake are as shown in Figure 5 and 
Table I. A significant reduction of uptake is shown in 60% sea water as com- 
pared to 9S% sea water in the tests, both at 10 and 20 C. 

The mean hourly water exchange fractions (K), adjusted for a wet weight of 
10 g are given in Table II, together with O 10 values, estimated as before. If we 
exclude the values from 95 % SW because of the questionable validity of the group 
tested at 20 C, the value of 1.63 may be taken as the Q 10 of THO uptake. 
This corresponds well with the value of 1.61 obtained for DHO in 60% SW. 



242 



RALPH I. SMITH AND PAUL P. RUDY 



(d.) Comparison of DHO and THO uptake 

In three groups out of four, the THO method resulted in lower uptake values 
than were obtained by the DHO method (Fig. 4, 5). Both methods showed a 
significant reduction in water exchange at the lower salinity, with the exception 
of the DHO, 20 C, pair. Using the slopes of the other three groups as represent- 
ing the actual relationship between salt concentration and water turnover, it would 
appear that the uptake determined by the DHO method in 95% SW at 20 
out of line and may be rejected. 



is 



100 
90 
80 

70 
60 

50 
40 

3 30 
I " 

LO 2 

.E ' 5 

o 100 

'-= 90 

80 

3 70 

(/) 60 
50 

40 

I 30 
25 

20 
15 



10 



T r 



1 r 



DHO- 
THO 



A. 60 % Sea water 




20C 



IOC . 



B. 95 % Seawater 




20C 



IOC 



4 5 6 7 8 9 10 20 

Wet weight in grams 



30 40 



60 80 100 



FIGURE 4. Comparison of uptakes of DHO and THO by H. nudus in respect to salinity, 
temperature, and wet weight, to show general smiliarity. 

The ratios of K (THO)/K (DHO) derived from the other 3 groups average 
0.94, that of the aberrant group being 1.01. The "t" indicates no significant dif- 
ference between THO and DHO uptakes (as % saturation adjusted to 10 g wet 
weight) obtained in 95% SW at either 10 or 20 C (Table I). In 60% SW, 
significance is indicated by a probability greater than 0.05 at 20 C, and less than 
0.02 at 10 C. The lack of significance within some groups may reflect low 
values (41 or less). 



"n" 



WATER-EXCHANGE IN CRAB HEMIGRAPSUS 



243 



However, when the ratio of THO/DHO uptakes is calculated for all paired 
determinations, involving all 4 groups, totaling 133 animals, the mean uptake of 
THO is 95.98% that of the DHO uptake. Even including the group tested in 
95% SW at 20 C, which was "out of line," this value of 96% is significantly dif- 
ferent from 100% at the 1% level of probability (99% confidence limits), and it is 
concluded that, in our experiments, the measured uptake of THO is significantly 
lower than that of DHO. 



13 
C 

'e 

LT> 



30 



25 



C 

o 

15 

rs 

15 
co 



20 



15 



I I 
DHO THO 



- 2 Std. errors 




J_ 



50 



60 70 80 

Percent Seawater 



90 



100 



FIGURE 5. Uptake of DHO and THO by PL nudiis as a function of salinity and tempera- 
ture, adjusted for a wet weight of 10 grams. Blocks indicate 2 standard errors above and 
below the means. 

DISCUSSION 

But the actual meaning of this "significantly lower" uptake of THO in relation 
to DHO is not so easily determined. That the procedures used gave a small dif- 
ference is reasonably clear. It is equally clear that the previously-reported differ- 
ence of 20% (Smith, 1970) is not substantiated. Further refinement of procedures 
would probably reduce the scatter evident in the present results in particular, 
increase of the exposure time so as to achieve saturations of 50% would be 
desirable. The 15-minute exposure was selected on the assumption that Hemi- 
(jraf>sns audits would have a permeability comparable to that of Carcinus, but it 
proved to be less permeable than expected (Carcinus: K (DHO) : = 2.07 in 50% 
SW at 18 C, Smith 1970; H. nudiis: K (DHO) = = 1.34 in 60% SW at 20, this 
study). 

It is possible that the difference observed between DHO and THO uptake 
lay in some aspect of the distillation procedures : possibly giving a spuriously high 
value for DHO expressing itself in the density determinations, possibly giving a 
spurious low value for THO as a result of undetected contamination (dilution) 
with atmospheric water in the vacuum distillation. 



244 RALPH I. SMITH AND PAUL P. RUDY 

An attempt was made to make a second determination of DHO upon the 
vacuum-distilled samples, but this was unfortunately delayed for some weeks and, 
because many of these samples were contaminated in such a way as to raise their 
density, the results had to be discarded. The evidence for an isotope effect is not 
conclusive. We can conclude that the methods of the respective authors for DHO 
and THO determinations have yielded differing results suggestive of an isotope 
effect. But while it may be expected that adherence to a single method of distilla- 
tion may make our procedures more alike, the fact that different final steps must 
be used to determine concentrations of the radio-active THO and the non-radio- 
active DHO makes conclusive demonstration of an isotope effect unattainable by 
our methods. 

Despite this difficulty, we have in fact obtained different measures of water 
flux on the same experimental animal and, if we accept the possibility of an isotope 
effect, it follows that the actual flux of H 2 O may be higher than that indicated 
by heavier isotopes. If a mass difference of 3:2 (T:D) results in fluxes in the 
proportions of 96:100, then a mass difference of 2:1 (D:H) might be expected 
to produce a difference in fluxes as great as 100: 105.3. In other words, the relative 
uptakes of THO, DHO, and H 2 O might be as ca. 91 :95 : 100. 

These differences in uptake as indicated by different isotopic tracers are not 
great, but may partly explain why measured values of urine production in crabs 
have in some cases been greater than those predicted from water-exchange mea- 
surements, as, for example, the urine volumes measured in Carcinus in 30% SW 
by Shaw (1961) and Binns (1969), as compared to the net influx values cal- 
culated on the basis of THO exchange (Rudy, 1967) and DHO exchange (Smith, 
1970). In Table III (line 11) is shown a calculation of the daily net influxes 
of water at 20 C (presumed equal to urine volume) in Hemigrapsus nitdus, 
calculated on the above assumption that uptakes of THO:DHO:H,O are in the 
proportions of 91:95:100. These values are less than comparable values reported 
for Carcinus (line 12), indicating that Hemigrapsus nudus has a lower net water 
influx and should produce less urine than Carcinus. The values for net water 
influx in Carcinus in 60% SW as previously determined by Smith (DHO) and 
Rudy (THO) are caluculated and shown in line 12 (Table III), together with 
urine volume as estimated by H 2 O-clearance methods by Shaw (1961). These 
values show an overall inverse relation to isotope weight consistent with the pres- 
ent hypothesis of an isotope effect. This trend is less clear in the case of 95% SW, 
but it may be noted that the net influx value from Rudy (THO) and urine volume 
from Shaw (clearance) had to be obtained by interpolation. The results are only 
consistent with the concept of an isotope effect ; they do not prove it. What they 
show is that the THO method for some reason tends to indicate a low value for 
water flux relative to the DHO method, and that measures of urine volume in crabs 
by methods not involving the heavier isotopes of water may yield higher values. 
However, the difference between urine volumes as determine! by methods not in- 
volving water isotopes, and the net influxes calculated from water-isotope exchange 
studies, is still too great to be explained solely on the basis of an isotope effect as 
small as the 5-10% suggested here. Such factors as unstirred layers or pores may 
also be involved. And, obviously, studies by several methods used upon the same 
species should furnish more satisfactory data than is currently available. 



WATER-EXCHANGE IN CRAB HEMIGRAPSUS 



245 



TABLE III 

(Lines 1-11) Calculation of water influxes in Hemigrapsus nudus at 10 C in 60% and 95% SW 

according to hourly water exchange (K) values based upon simultaneous THO and DHO 

uptake, with estimates of net H 2 influx. Water content assumed to be 70%. 

(Line 12) Published comparable values for Carcinus 



1. Medium, % SW 
and cone, millios- 
moles 


60 (600 millismoles) 


95 (950 millismoles) 


2. Concentration of 
blood, millios- 
moles 


845 


965 


3. Cone, of blood as 
%SW 
4. Mole fraction 
water of medium 
5. Mole fraction 
water of blood 
6. Mole fraction 
difference 


84.5 
0.9893 
0.9850 
0.0043 


96.5 
0.9832 
0.9829 
0.0003 


7. Hourly water ex- 
rliange fraction 
(K) at 10 C 


THO 


DHO 


H 2 O (est.) 


THO 


DHO 


H 2 O (est.) 


0.76 


0.83 


ca. 0.855 


0.94 


0.97 


ca. 1.025 


8. Daily water influx 
(K X 70 X 24) as 
% body weight 
per day 


1276.8 


1394.4 


1436.4 


1579.2 


1629.6 


1722.0 


9. Daily net water 
influx as % of 
total influx 


0.435 


0.435 


0.435 


0.0305 


0.0305 


0.0305 


10. Daily net influx as 
% of body weight 
at 10 C 


5.55 


6.06 


6.25 


0.48 


0.50 


0.525 


11. Daily net influx in 
h. nudus at 20 C 
(Q.o == 1.62) 


8.99 


9.82 


10.1 


0.78 


0.81 


0.85 


12. Calculated daily 
net influx in 
Carcinus (DHO 
and THO) and * 
urine volume 
(direct meas.) 


net influx 
9.4 
Rudy 
(1967) 
recalc. 20 


net influx 
14.5 
Smith 
(1970) 
recalc. 18 


urine vol. 
16.5 
Shaw 
(1961) 
16 


net influx 
1.25 
Rudy 
(1967) 
recalc. 20 


net influx 
1.19 
Smith 
(1970) 
recalc. 18 


urine vol. 
5.0 
Shaw 
(1961) 
16 



SUMMARY 

1. The crab Hcuiiyrapsus midus regulates the osmotic pressure of its blood in 
media down to less than 60% seawater, and is iso-osmotic in 100% seawater and 
higher salinities. 



246 RALPH I. SMITH AND PAUL P. RUDY 

2. Measurements of simultaneous uptake of tritiated water (THO) and deuter- 
ated water (DHO) give uptake values for THO about 96% those obtained with 
DHO. 

3. The Q 10 of uptake of both isotopes is about 1.62, and the relation of uptake 
of both to body weight is similar. 

4. The results are consistent with, but do not prove, the concept of a small 
isotope effect in the uptake of THO and DHO. Published reports of higher water 
fluxes based on methods not involving isotopes of water are consistent with the 
argument for an isotope effect. It is suggested that water fluxes based on methods 
using THO:DHO:H.,O as ordinarily employed are of the relative magnitudes 
91:95:100. 

LITERATURE CITED 

BINNS, 1\., l ( '(i ( A The physiology of the antennal glands of Carciniis IIIUCIHIS (L.). II. Urine 

production rates. /. Exp. Biol., 51 : 11-16. 
CHINARD, F. P., AND T. ENNS, 1954. Relative rates of passage of deuterium and tritium oxides 

across capillary walls in the dog. Amcr. J. Physiol., 178 : 203-205. 
ENNS, T., AND F. P. CHINARD, 1956. Relative rates of passage of H 1 H S O 18 and of HMD 18 across 

pulmonary capillary vessels in the dog. Am. J. Physiol., 185 : 133-136. 
JOHNSON, P. A., AND A. L. BABB, 1956. Liquid diffusion of non-electrolytes. Chevn. Rev., 56: 

387-453. 

KAVANAU, J. L., 1964. U'atcr and Solute-Water Interactions. Holden-Day, Inc. San Fran- 
cisco, 101 pp. 
I'ACA. \KLLI, C. V., AND A. K. SOLOMON, 1957. The rate of exchange of tritiated water across 

the human red cell membrane. /. Gen. Physiol., 41 : 259-277. 
PARRY, G., 1955. Urine production by the antennal glands of Palaemonetes varians (Leach). 

/. Exp. Biol, 32 : 408-422. 
PIMENTEL, G. C., AND A. L. McCLELLAN, 1960. The Hydrogen Bond. W. H. Freeman, San 

Francisco, 475 pp. 
RUDY, P. P., JR., 1967. Water permeability in selected decapod Crustacea. Coinp. Biochcui. 

Physiol., 22 : 581-589. 
SHAW, J., 1961. Studies on ionic regulation in Curcinus macnas (L.). I. Sodium balance. 

/. Exf. Biol., 38 : 135-152. 
SMITH, R. I., 1967. Osomotic regulation and adaptive reduction of water-permeability in a 

brackish-water crab, Rhithropanopcus harrisi (Brachyura, Xanthidae). Biol. Bull.. 

133: 643-658. 

SMITH, 1\. I., 1970. The apparent water-permeability of Carcimts macnas (Crustacea, Brachy- 
ura, Portunidae) as a function of salinity. Biol. Bull., 139: 351-362. 
WANG, J. H., C. V. ROBINSON AND I. S. EDELMAN, 1953. Self-diffusion and structure of liquid 

water. III. Measurement of the self-diffusion of liquid water with H 2 , H 3 , and O 1S 

as tracers. J. Amcr. Chcm. Soc., 75 : 466-470. 
WELSH, J. H., R. I. SMITH AND A. E. KAMMER, 1968. The estimation of D 2 O in water or 

blood. Pages 184-188 in Laboratory Exercises in Invertebrate Physiology. Burgess 

Publishing Co., Minneapolis. 



Reference : Biol. Bull., 143: 247-255. (August, 1972) 



A COMPARISON OF IN SITU AND IN VITRO RESPONSES 
OF CRUSTACEAN HEARTS TO HYPOXIA * 

DANIEL F. STIFFLER2 AND AUSTIN W. PRITCHARD 
Department of Zoology, Oregon State University, Corvallis, Oregon 97331 

Although crustacean cardiac physiology has been extensively researched, there 
remain several aspects which have scarcely been investigated. One such area has 
been the response of the crustacean heart to deficiencies in oxygen. 

An early indication of the cardiac response to hypoxia was noted by Burger and 
Smythe (1953) in the lobster Homarus americanus. It was observed that the heart 
of this animal slowed its rate of beating when the lobster was out of the water. 
In this situation the gill filaments would be collapsed and the effective exchange 
area reduced, thereby placing the animal in an hypoxic situation. Larimer (1962, 
1964a, 1964b), in a series of studies on the crayfish Procambarus siniulans, found 
that these animals demonstrate a marked bradycardia when the oxygen is driven 
from the water containing them. Thompson and Pritchard (1969) subjected the 
burrowing shrimp Callianassa californiensis to hypoxia and noted a significant 
reduction of heart rate at very low oxygen concentrations. 

In the present investigation the response to hypoxia of the heart of the crab 
Cancer inagister (Dana) will be described. Previous investigations of these re- 
sponses have been limited to the response of the In situ heart. This report will 
also deal with the response of the isolated heart to hypoxia. The effect of lowered 
oxygen concentrations on amplitude or magnitude of contraction is also examined. 

METHODS AND MATERIALS 

This study was carried out at the Oregon State University Marine Science 
Center at Newport, Oregon and at the main campus in Corvallis. 

Adult specimens of Cancer magistcr ranging in carapace width from 10 to 18 cm 
were collected from Yaquina Bay and maintained at 12 C. 

All experiments were performed at 12 1 C which approximates the tempera- 
ture in the area from which the animals were collected. 

Recording of heart rate and amplitude of contraction was achieved by the use 
of a Narco Bio-Systems physiograph in conjunction with a Narco Type B photo- 
electric force transducer-type myograph. 

Oxygen concentration in the experimental containers was manipulated by bub- 
bling nitrogen or air directly into the water or perfusion solution (in the case of 
the isolated hearts). A Yellow Springs Instrument Company Model 54 oxygen 
meter and Model 5420 oxygen probe were used to moniter the oxygen concentra- 
tion in the testing bath. 

1 Supported in part by Institutional Sea Grant GH-45. 

: This work was submitted by Daniel F. Stiffler to the Graduate School at Oregon State 
University in partial fullfillnicnt of the requirements for the Master of Science degree. 

247 



248 DANIEL F. STIFFLER AND AUSTIN W. PRITCHARD 

Ani?r>ais were prepared for /;/ situ recording by inducing autotomy of their legs 
and then tied to a restraining board. Limb autotomy seemed to have no effect on 
the pattern of the recorded heart beat, as demonstrated by recordings of the heart 
beat of a few animals -with intact legs. After the removal of a small piece of 
carapace overlying the heart, a bent pin was inserted into the still intact hypodermis 
and connected by a thread to the myograph. The oxygen probe and air stones 
were then positioned in the bath and the container was covered to restrict the 
entry of air. 

The preparation for the in vitro heart beat recordings involved a modification 
of a method described by Welsh and Smith (1960). The pericardial cavity was 
carefully exposed and ligatures were tied around the posterior lateral ligaments of 
the heart. These later served as a convenient means of handling the heart. Next, 
the abdominal artery was cannulated with a piece of suitable sized polyethylene 
tubing. Finally the anterior arteries were ligatured and the heart was removed to 
the recording chamber and attached to the myograph. The recording chamber con- 
sisted of a 4 ) 4x6 inch plastic refrigerator container equipped with air stones 
and an oxygen probe. The bottom of the chamber was pierced by a tube through 
which the perfusion solution passed and to which the cannula was attached. 

After mounting the heart in the chamber, the rate of perfusion could be con- 
trolled by means of a Teflon needle valve. Shortly after initiating perfusion the 
heart resumed its rhythmic beat with a steady rate and amplitude. Before a prep- 
aration was judged usable it had to meet the following criteria: display of reason- 
ably steady rate and amplitude; recovery to near normal rate and amplitude after 
hypoxic stress ; and response to changes in perfusion rate with corresponding 
changes in rate and amplitude. An increase in perfusion rate has been shown to 
result in an increase in rate and amplitude for the heart of Maia sqitinado 
(Izquierdo, 1931). 

All hearts were perfused with a solution identical to that used by Davenport 
(1941). 

The same protocol was used for both in situ and isolated hearts in determining 
the response to hypoxia. After the recording instruments were attached the animal 
was allowed to stabilize at least ten minutes and the oxygen concentration of the 
water was recorded. Nitrogen was then passed through the water for a short 
period. After the heart beat and oxygen concentration had again stabilized the 
procedure was repeated. This was done until the oxygen concentration had de- 
creased to below 1 mg O 2 per liter of sea water. Air was then re-introduced into 
the bath and the heart beat was allowed to return to normal. During the recovery 
period the oxygen concentration was periodically recorded in order to evaluate 
the latency of the response to oxygen. 



RESULTS 

The heart rates of the crabs were found to vary somewhat from animal to 
animal but were fairly consistent from one experiment to the next for a given 
animal. The mean rate for in situ hearts when beating at maximum was 79 beats 
per minute with a range of 72 to 92. The mean rate for isolated hearts at maxi- 
mum was 54 beats per minute with a range of 37 to 81. 



HYPOXIA AND CIRCULATION IN CRABS 



240 



Since this investigation is primarily concerned with relative values the data are 
expressed as a per cent of maximum rate. To prevent the possibility of random 
accelerations of short duration from biasing the data, the maximum rate had to 
be sustained for a period of at least three minutes to be considered as valid. The 
resultant values arc- best termed relative rate. 

The amplitude recorded by the myograph provides a rough index of the 
strength of contraction of the hearts. This is quantifiable in terms of relative 
amplitude. In some of the recordings amplitude varied slightly from contraction 
to contraction but was maintained at an overall consistent level. This was espe- 
cially so in the case of the in situ hearts. To determine the relative amplitude 
under these conditions, the- heights of ten consecutive peaks, chosen at random. 
were averaged. The myographic recording of the amplitude of contraction in 
in situ hearts is complicated by the complex attachments of the heart, both within 



100 



75 



E 
=> 
6 

X 

o 



50 




01 234567 

Oxygen Concentration (mg 2 / I s.w.) 

FIG r UK 1. Relationship hetwcen heart rate () and amplitude (O) and oxygen concen- 
tration for in situ hearts. Each curve represents the results of eight experiments in which 
rate and amplitude were recorded simultaneously. The rates and amplitudes are expressed in 
terms of per cent of maximum while the oxygen concentration is in nig O/l sea water. The 
standard errors ranged from 0.8^ to 6.0% for rate and from 2.0% to 8.1% for amplitude. 

the pericardial cavity and externally with the myograph. The attachment of the 
heart to the myograph in the /;/ situ state is, of necessity, indirect due to the need 
to preserve integrity of the open circulatory system. To accomplish this the pin 
connected to the myograph was inserted in the hypodermis which, in turn, was 
indirectly connected to the heart. This left the open circulatory system intact but 
resulted in a complex transmission of the heart's action to the myograph. This did 
not affect the recording of rate but may have significantly altered the pattern of 
recorded amplitude. With the isolated hearts the myograph was connected directly 
to the hearts themselves. Since the amplitude response of the isolated hearts was 



250 



DANIEL F. STIFFLER AND AUSTIN W. PRITCHARD 



similar in nature to that of the in situ hearts the greater variability of the latter 
may have been due to the indirect means of recording. 

A persistent feature of many in situ recordings was the occasional appearance 
of a transient cardiac arrest. This was observed to occur both in diastole ( Fig. 3a) 
and in systole and was often seen either accompanying or just preceding limb 
or limb stub movement. Similar cardiac inhibition has been reported in Asellus 
aquatic us, an isopod (Needham, 1954), Panuliriis argns, a lobster (Maynard, 
1960) and Procainbams clarkii. a crayfish ( Larimer and Tindel, 1966). 

The responses of both /;/ situ and isolated hearts to hypoxia were marked 
bradycardia and depression of amplitude. Although the responses were similar in 
most respects, certain differences were apparent. Figure 1 depicts the response of 
the in situ hearts to hypoxia and it may be seen that as oxygen concentration fell 
below 1 mg O 2 /l both rate and amplitude decreased to less than 50 per cent of the 
values in air-saturated water. The pattern of this decrease, however, is different 
for rate and amplitude. "While the decrease in rate is clearlv hyperbolic, with the 
most significant decrease occurring between 1 and 2 nig O 2 /l, the decrease in ampli- 
tude tends more toward linearity in this range of oxygen concentrations. The pat- 
terns of decrease in rate and amplitude of isolated hearts, however, are very much 
alike, both tending toward a hyperbolic relationship (Fig. 2). It is felt that the 



100 



. 75 



50 








01 2345678 

Oxygen Concentration ( mg 02 /I s.w. ) 

FIGURE: Relationship between heart rate () and amplitude (O) and oxygen con- 
centration for isolated hearts. Each curve represents the results of six experiments in which 
rate and amplitude were recorded simultaneously. The rates and amplitudes are expressed in 
terms of per cent of maximum while the oxygen concentration is in mg O/l sea water. The 
standard errors ranged from 0.6% to 8.0% for rate and from 0.6% to 5.4% for amplitude. 

indirect means of recording in situ heart movements is responsible for the appar- 
ently anomalous pattern of in situ amplitude. 

The time course of the recovery of the hearts to near the maximum rate upon 
readmission of oxygen to the water after hypoxic stress differed greatly in in situ 
and isolated hearts. Nineteen experiments were performed and in each case the 



HYPOXIA AND CIRCULATION IN CRABS 



251 



response of the in situ hearts to oxygen was much more rapid than was the re- 
sponse of the isolated hearts. The mean recovery time for in situ hearts was 
22 seconds while the mean recovery time for isolated hearts was 127 seconds. These 
are significantly different at the 0.001 level (t-test). The mean rates of increase 




7.9 mg/ 



5 min. I 



2.5 mg/ | 



- 8 m ' n ^-^JJJJJJJJJJJJJJJJJJJJJ. 



Z.Omg/i 



0.8 mg/ 




7.6 mg/l 



I.I mg/| 



Airj, 




. 20 sec. 



5.8 mg/ | 



FIGURE 3. Recordings of typical ;';/ situ (A) and isolated (B) heart beats. The records 
are continuous except where breaks are indicated. Oxygen concentration at various points is 
given in mg Os/l sea water. 

of oxygen concentration were roughly similar in the two situations being 1.82 mg 
Oo/l/min for in situ and 1.24 mg O 2 /l/min for isolated preparations. Typical 
recordings showing these responses are given in Figure 3. 

As a precaution against the possibility that the response of the /;/ si fit hearts 
to aeration of hypoxic water may have been a mechanoreceptive one, associated with 



252 



DANIEL F. STIKN.KK AND AUSTIN W. 1'RITCHAKD 



the turbulence caused by aeration, several crabs were subjected to sudden vigorous 
nitrogen bubbling while under hypoxic stress (Fig. 4). In no case was there a 
duplication of the aeration response. 



l 

mu r uWVJVH \ifiu llTilP '; I) 
I 

0.7 mg/| 




mg/| 




2.0mg/| 



20 sec, i 



FIGURE 4. Continuous recording showing the failure of the in situ heart to respond to 
turbulence caused by vigorous nitrogen bubbling. The duration of aeration and Migration bub- 
bling is shown above recording while oxygen concentration of the water is indicated below. 

I )ISCUSSION 

The variation in the maximum heart rate probably results from a number of 
factors. Since the animals were held for varying lengths of time without feeding, 
their nutritional states might have varied. More importantly, different levels of 
"trauma" following preparation of the animals undoubtedly had some effect on 
the level of heart rate and amplitude. It is felt, however, that since these param- 
eters were quite stable within a given preparation, and since relative rates and am- 
plitudes were used, the variability floes not significantly alter the interpretations of 
the data. 

A comparison of the mean maximum heart rates of the isolated and /;; sit it 
hearts shows that the latter beat much more rapidly ( 79 min i's. 54/min). The 
difference may lie due to the absence of the cardio-acceleratory nerves and pos- 
sibly the pericardial organs in the isolated preparations. The pericardial organs 
are neurosecretory elements which have a pronounced cardio-excitatory effect 
(Cooke, 19(4). 

The bradycardia resulting from hypoxia confirms that observed in other crusta- 
ceans investigated. These include Procanibanis sinnilaiis (Larimer, 1962, 1964a, 
1964b) and Callainassu californiensis (Thompson and Pritchard, 1969). 

The significance of the hypoxia-induced bradycardia is not clear. Larimer 
(1962) felt that it might be due to a high sensitivity of the heart muscle to lowered 
oxygen concentration. Subsequent work (Larimer, 1964a), however, indicates 
that this may not be the case, at least for P. siinnhins, as the per cent extraction of 
oxygen from the respiratory stream increased during hypoxia. To explain this 



HYPOXIA AND CIRCULATION' IN CRABS 253 

Larimer has hypothesized an increased rate of circulation, facilitated by an in- 
creased stroke volume and decreased peripheral resistance during hradycardia. A 
similar increased per cent extraction of oxygen has been reported for the lobster 
Homarus vulgaris during hypoxia (Thomas, 1954) and circulatory responses were 
also postulated as an explanation. 

The increased circulation rate at lo\v concentrations hypothesized by Larimer 
(1964a) might be advantageous for an animal such as P. siniiilans. whose respira- 
tory pigment has a high affinity for oxygen. Larimer and Gold (1961) report a 
P r , of 3.5 mm Hg for this crayfish. Cancer niai/ister, however, has a low oxygen 
affinity hemocyanin with a P- of 19.6 mm Hg at 10 C (Johansen, Lenfant and 
Mecklenburg, 1970). Callianassa- calif orniensis also has a high oxygen affiniu 
hemocyanin with a P- (1 of 3-4 mm Hg at 10 ( Miller and Pritchard, unpublished 
data), and shows a bradycardia at low oxygen concentrations (Thompson and 
Prithcard, 1969). Data regarding changes in per cent extraction of oxygen for 
Cancer ina</is/cr during hypoxia are not available. However, Johansen ct al. 
( l c ^70) report an increase in the gradient between exhaled water and arterial blood 
oxygen during partial hypoxia which might reflect a decreased per cent extraction 
of oxygen from the respiratory stream. This, however might also be explained by 
a possible increased ventilation in these animals during hypoxia (Johansen ct al.. 
1970; Stiffler, 1970). 

Cancer nui</istcr is capable of regulating its metabolic rate over a wide range of 
oxygen concentrations (Johansen ct a/., 1970). It is noteworthy that the lower 
limits of this regulation correspond roughly to the range of oxygen concentrations 
in which the decrease in heart rate and amplitude, reported here, are most apparent. 
Regulation of metabolic rate over a wide range has also been observed in the 
shrimp C. californiensis (Thompson and Pritchard, 1969). Bradycardia in this 
species also occurred at oxygen concentrations roughly corresponding to the critical 
oxygen concentration at which metabolic rate began to decrease. These authors 
suggested that the maintenance of a constant heart rate aids this animal in regulat- 
ing its oxygen consumption rate. 

The decrease in amplitude of contraction observed in this study is difficult to 
interpret. The fact that it occurred consistently in isolated hearts as well as in situ. 
hearts would seem in indicate that it is not an artifact of pin placement in the latter 
case. Although the amplitude of contraction is certainly not a reliable index of 
cardiac output, changes in this parameter may roughly indicate changes in stroke 
volume. If this is the case the increased stroke volume hypothesized by Larimer 
(1964a) for the crayfish does not appear to be operating in the case of Cancer 
niat/ister. What may be happening is that as the oxygen concentration decrease>> 
below the range where the animals regulate metabolic rate and oxygen consumption 
declines, the circulation rate also decreases. The fact that the isolated hearts 
showed a bradycardia under hypoxic conditions indicates that the heart muscle may 
be sensitive to oxygen deprivation in this animal. 

All of this suggests that an increased rate of circulation during hypoxic stress 
as proposed by Larimer (1964a) for crayfish is not a generalized response for 
decapod crustaceans. The present data, as well as that of Johansen ct al. ( 1970 I 
and Thompson and Pritchard ( 1969), suggest that the bradycardia may be accom- 
panied by a decrease in circulatory rate in at least some decapods. In any case 



254 DANIEL F. STIFFLER AND AUSTIN W. PRITCHARD 

the interactions involved in gas exchange cannot be fully assessed for Cancer 
inac/ister until measurements of cardiac output and per cent extraction of oxygen 
from the respiratorv stream have been made under conditions of varying oxygen 
concentration. 

The response of the isolated hearts to hypoxia was quite similar to that of 
the in situ hearts. The correlation between changes in amplitude and rate was 
much closer in the case of the isolated hearts, however. For the in situ hearts 
the relationship between heart rate and oxygen concentration was clearly hyperbolic, 
whereas the relationship between amplitude and oxygen concentration tended more 
toward linearity (Fig. 1 ). This might be explained by the indirect connection be- 
tween the heart and the myograph discussed above. With the isolated hearts, both 
functions tended toward a hyperbolic relationship (Fig. 2). This might be ex- 
pected with the more direct means of recording the actions of the isolated hearts. 

One of the more interesting differences between the isolated and in sitit hearts' 
responses lies in their respective patterns of recovery. "While the in situ hearts re- 
covered quite rapidly, within a few seconds of aeration of hypoxic water, the isolated 
hearts were quite slow to recover, consistently taking several minutes to do so 
(Fig. 3a and 3b). As there is no way of instantaneously restoring the oxygen to 
the water the dead time involved in aeration may be significant. 

A possible explanation of the discrepancy in these latencies is that the /;; sitit 
heart remained under the control of the animal's nervous system. This would be 
suggestive of a receptor or receptors sensitive to oxygen. Such hypothetical re- 
ceptors have been implicated in the responses of other arthropods to oxygen 
( Waterman and Travis. 1953: Larimer, 1964a : Farley and Case, 1969; Gamble, 
1971). The possibility that the response might be due to the turbulence associated 
with aeration can be ruled out as vigorous bubbling of nitrogen, initiated suddenly 
in still, hypoxic water, did not elicit the response (Fig. 4 ). 

Although the idea of oxygen receptors is an intriguing one, until such receptors 
are found and action potentials recorded from their nerves at varying oxvgen con- 
centrations, their existence must remain highly speculative. 

SUMMARY 

1. Heart rate and amplitude were recorded for both in situ and isolated hearts 
of Cancer inagistcr exposed to lowered oxygen concentrations. 

2. Both rate and amplitude declined markedly as the oxygen was driven from 
the water. This occurred in both in situ and isolated hearts. 

3. The recovery of in situ hearts to near normal rate and amplitude occurred 
quite rapidly upon the readmission of oxygen to the water. 

4. Isolated hearts recovered very slowly to aeration of hypoxic water. 

LITERATURE CITED 

BURGER, W. J., AND C. McC. SMYTHE, 1053. The general form of circulation in the lobster 

Hoinants. J. Cell. Coinp. Physio!., 42: 369-383. 
COOKE, I. M., 1964. Electrical activity and release of neurosecretory material in crab peri- 

cardial organs. Coip. Bioclicin. Physiol., 13: 353-366. 
DAVENPORT, D., 1941. The effects of acetylcholine, atropine, and nicotine on the isolated heart 

of the commercial crab, Cancer inaglstcr Dana. Phvsiol. Zool., 14: 178-185. 



HYPOXIA AND CIRCULATION IN CRABS 255 

FARLEY, R. D., AND J. F. CASE, 1968. Perception of external oxygen by the burrowing shrimp 

Callianassa californiensis Dana and C. affinis Dana. Biol. Bull., 134: 261-265. 
GAMBLE, J. C., 1971. The responses of the marine amphipods Corophium arenarium and C. 

volutator to gradients and to choices of different oxygen concentrations. /. E.\-[>. Biol., 

54 : 275-290. 
IZQUIERDO, J. J., 1931. A study of the crustacean heart muscle. Proc. Roy. Soc. London 

Series B, 109 : 229-250. 
JOHANSEJST, K., C. LENFANT AND T. A. MECKLENBURG, 1970. Respiration in the crab, Cancer 

magistcr. Z. Vergl. PhysioL, 70 : 1-19. 
LARIMER, J. L., 1962. Responses of the crayfish heart during respiratory stress. Ph\siol. Zool., 

35: 179-186. 

LARIMER, J. L., 1964a. The patterns of diffusion of oxygen across the crustacean gill mem- 
branes. /. Cell. Comp. PhysioL. 64 : 139-148. 
LARIMER, J. L., 1964b. Sensory induced modifications of ventilation and heart rate in crayfish. 

Comp. Biochem. PhysioL, 12 : 25-36. 
LARIMER, J. L., AND A. H. GOLD, 1961. Responses of the crayfish, Procainbanis siiniilans, to 

respiratory stress. PhysioL Zool., 34: 167-176. 
LARIMER, J. L., AND J. R. TINDEL, 1966. Sensory modifications of heart rate in crayfish. 

Animal Behaviour, 14: 239-245. 
MAYNARD, D. M.. 1%(). Heart rate and body size in the spiny lobster. Plivsinl. Zool., 33: 

241-251. 

NEEDHAM, A. E., 1954. Physiology of the heart of Ascllus aquations L. Nature, 173: 272. 
STIFFLER, D. F., 1970. Cardiac and respiratory responses to hypoxia in the crab, Cancer 

magistcr (Dana). Master's thesis, Oregon State University, 42 pp. 
THOMAS, H. J., 1954. The oxygen uptake of the lobster (Hoinants znilgaris Edvv.). /. E.vp. 

Biol.,31: 228-251. 
THOMPSON, R. K., AND A. W. PRITCHARD, 1969. Respiratory adaptations of two burrowing 

crustaceans, Callianassa ea/ifoniiensis and Upogcbia pugettensis (Decapoda, Thalas- 

sinidea). Biol. Bull., 136 : 274-287. 
WATERMAN, T. H., AND D. F. TRAVIS, 1953. Respiratory reflexes and the flabellum of Limulus. 

J. Cell. Comp. PhysioL, 41 : 261-290. 

WELSH, J. H., AND R. I. SMITH, 1960. Laboratory Exercises in Invertebrate Physiology. 
[Rev. ed.| Burgess Publishing Company, Minneapolis, 179 pp. 



Ket'erence : iliol. Hull., 143: 256-2o4. (August, l ( >72) 



ACTION OF HYDROSTATIC I'KFSSURF ON SEA I RC11IX CILIA 3 

PAUL G. YOUXC,.- A. DOROTHY YOUXC AND ARTHUR M. XIMMKRMAX 

Hcfiiirtnicnt at Zooloi/y. I'liri'd'sity of I ormito, 1 orotito, ('<;<;</</ dinl 
Mtirin,' Biological Laboratory, U'otnls Hole. Massachusetts 

Hydrostatic pressure has been shown to atiect organized cellular structures and 
macromolecular svnthesis (Zimmerman, 1970). In several different: species of 
protozoa (Kitching, 1957, 1970) and in marine tissue ( Flugel and Schlieper. 1970), 
it has been reported that ciliary activity is inhibited by pressure. Pressure causes 
the disappearance of microtubular arrays in structures such as the mitotic apparatus 
( /immerman and Marsland, U'M; Zimmerman and Philpott, unpublished) and 
the axopodia of Actinosphaerium ( Tilney, Hiramoto and Marsland. l c >(><>). In 
Tetrahymena the proximal portions of the central ciliary fibers and longitudinal 
microtubules are affected bv pressures of 7500-10,000 psi ( Kennedv and Ximnier- 
man. 1970). However, line structural analysis of the cilia and sperm rlagellae of 
sea urchin embryos indicate that these structures are resistant to pressure treatment 
(Tilney and Gibbins. 190S. 19(,q : Marsland. 1970). 

In protozoa, protein synthesis is essential for complete flagellar and cilia re- 
generation (Rosenbaum and Child. 1 ( H>7; Kosenbaum, Moulder and Ringo. I'Xi'h ; 
however, RXA and protein synthesis are not necessary for initial cilia formation 
and regeneration in sea urchin embryo-- < Auclair and Meismer, 1965; Auclair and 
Siegel, 1966). The sea urchin thus provides a relatively simple system for study- 
ing processes involved in the organization and assembly of the cilium. 

The experiments reported here were designed to investigate the effects of hy- 
drostatic pressure on both the intact cilium and the regenerating cilium in the sea 
urchin embrvo. 

METHODS AND MATERIALS 

Fmbrvo> i if .Irlnicla pitnchtlata were maintained in natural seawater ; embrvos 
of Strongylocentrotus piirpuratits were kept in artificial seawater (Instant Ocean). 
Eggs and sperm were shed by KC1 injection or electrical stimulation (Harvey. 
1956). After insemination development was allowed to proceed at 18 C for 
both species. 

Deciliation was performed according to Auclair and Siegel (1966). Hypertonic 
seawater treatment (29.2 g XaCl/liter of seawater for 1-2 min ) was found to 
remove essentially all cilia. Following deciliation embryos were resuspended in 
normal seawater for regeneration to occur. 

The initial stages of regeneration were observed and measured after staining 
with Lugol's iodine. Measurements on cilia longer than 6-S /j. were made on living 

1 Work supported in part by Xational Research Council of Canada. 

2 Predoctoral Fellmv Xational Research Council of Canada. 

256 



PRESSURE STUDIES ON CILIA 257 

specimens (in 5 ( / ( methyl cellulose) using phase contrast optics. The longest ob- 
servable cilium on each of five embryos was measured and the values averaged. 

The temperature-control housing, pressure pump and microscope-pressure 
chamber were similar to those described by Marsland (1950). Following decilia- 
tion the embryos at the stage of swimming blastula or early prism w r ere transferred 
to a Lucite chamber which was then placed inside the main pressure vessel. In all 
regeneration experiments, the pressure (2000-10,000 psi, which is equivalent to 
139-680 atm, or 137.8 >: lO'-USQ x 10 r ' Newtons/nr or Pascals) was applied 3 min 
after deciliation. Non-pressurized control embryos were placed in similar chambers 
but remained at atmospheric pressure. The embryos were observed at magnifica- 
tions up to 600 X while the cells were under pressure. 

RESULTS 

Preliminary observations 

The pattern of behavioral changes induced by pressure on swimming embryos of 
Strongylocentrotus [>iir[>tiratus and Arbacia punctiilata is dependent upon both mag- 
nitude and duration of treatment. The changes induced at tin- higher pressures are 
quite clear whereas at lower magnitudes of pressure there is considerable individual 
variation within a single group. 

Strongylocentrotus embryos ( swimming blastulae ) were subjected to varying 
magnitudes of pressure (0000-10,000 psi) while under continuous observation. 
Within two minutes after compression to 10,000 psi, the embryos began to slow 
down, although they still maintained their normal spiralling motion. Following 
3-5 min of compression the embryos began to collect on the bottom of the pressure 
chamber where they rotated with one pole against the surface. The rate of spin- 
ning gradually slowed and was replaced by a very rapid vibratory motion. After 
7-8 min most of the embryos were vibrating, however a few embryos retained 
their spinning motion and a few were completely motionless. After 10-12 min 
essentially all of the embryos were motionless. 

When the pressure was lowered to 8000 psi the cells responded in a similar 
fashion to that found at 10,000 psi except that some motion was evident 15 min 
after compression. At 7000 psi it took considerably longer for the embryos to dis- 
play the previously described effects. After 10 min at 7000 psi most of the embryos 
were still swimming, albeit more slowly than normal, and only a few embryos had 
settled to the bottom of the chamber. By 20 min all of the embryos had settled 
and were rotating slowly. Thirty min after compression, the embryos were still 
rotating although they were beginning to show signs of disaggregation. For this 
reason, longer durations were not employed at this pressure level. With a further 
reduction in pressure to 6000 psi it required between 20 and 30 min for most of 
the embryos to settle on the bottom of the chamber. At 50-60 min after com- 
pression, most of the embryos were still rotating. It was felt that the extent of 
disaggregation limited the usefulness of experiments of longer duration than 60 min. 

In experiments in which the pressure was released while the embryos were still 
active (i.e., displaying some movement) an initial burst of activity followed imme- 
diately upon decompression. 



258 



P. G. YOUNG, A. D. YOUNG, AND A. M. ZIMMERMAN 



It was generally found that .-Irhacia embryos displayed similar behavior; how- 
ever, they were more sensitive to pressure treatment than Strongylocentrotus 
embryos. 

Pressure effects on sea urchin cilia 

From the above study it was evident that high pressure treatment resulted 
in a loss of cilia. In order to investigate this phenomenon, Strongylocentrotus 
embryos were subjected to pressure and following decompression they were sttidied 
by phase contrast microscopy. The number of cilia lost depended partly on the 
magnitude and partly on the duration of treatment. At pressures of 8000 psi and 
above more than 90 r i of the cilia were removed within 10 min ; at 7000 psi more 
than cSO'/f of the cilia were removed 30 min after compression. When the pres- 
sure was reduced to 6000 psi more than 50% of the cilia were lost in 45-60 min. 
The cilia were lost as apparently intact structural units and could be observed 
floating in the medium. The detached cilia had distended bulbous distal tips simi- 
lar to the cilia which remained on the embrvos. 



20- 



16- 



O 

'- Q 
u 



A. punctulata 



control 



10 min pressure 




20 min pressure 
30 min pressure 







40 80 120 160 

Time after deciliation (min) 



200 



FIGURE 1. Duration of pressure treatment and cilia regeneration in Arbacia punctulata. 
Sea urchin prismatic stage larvae were deciliated and subjected to 10,000 psi for 10, 20 and 30 
min of compression. At various times after decompression the lengths of the cilia were mea- 
sured. 



PRESSURE STUDIES ON CILIA 



259 



In a series of experiments at 10,000 psi, in which groups of embryos were 
treated for varying durations (2-10 min), it was found that a few cilia remained 
on those embryos still undergoing the vibratory motion and essentially all cilia 
were gone from those embryos in which all motion had ceased. 

In embryos treated for long durations (30-60 min) at lower pressures (6000 
psi) the apical tuft cilia were found to be more resistant to pressure than other 
cilia. In embryos displaying partial cilia loss, the deciliation frequently occurred 
in a patch near the vegetal pole. 

Comparable studies with Arbacia. embryos indicated that they were more sensi- 
tive to pressure-induced cilia loss than Strongylocentrotus embryos. In some ex- 
periments, pressures as low as 2000 psi for durations of less than one hour caused 
essentially total loss of cilia from Arbacia embryos. 





control 



B 



pressure/20 min 



61 min after deciliation 

FIGURE 2. Pressure induced delay of cilia regeneration. The photomicrographs were taken 
61 min after deciliation by hypertonic seawater ; (A) Control embryo; (B) Experimental 
embryo was subjected to 10,000 psi for 20 min. Note the rounded appearance of the surface 
cells. Both control and experimental embryos were stained with Lugol's iodine. 

Pressure effects on cilia regeneration 

Since it is well established that high pressure interferes with microtubule polym- 
erization, the effects of pressure on cilia regeneration were investigated. The 
two parameters studied were the magnitude and the duration of pressure treatment. 
Embryos were deciliated by hypertonic sea water treatment and subjected to com- 
pression (2000-10,000 psi) for varying durations (10, 20, or 30 min). Following 
decompression, the lengths of the regenerating cilia were recorded and compared 
to controls. The regeneration delay for Arbacia embryos was found to be a func- 
tion of the duration of pressure treatment (Fig. 1). The onset of regeneration 
was delayed for 10-25 min in excess of the duration of compression. This delay 



260 



P. G. YOUNG, A. D. YOUNG, AND A. M. ZIMMERMAN 



was proportional to the duration of treatment. It was not possible to ascertain 
from this data whether or not the rate of cilia regeneration was affected by the 
pressure treatment. Photomicrographs of representative experimental (10,000 psi 
for 20 min ) and control embryos 61 min after deciliation are shown in Figure 2. 
The cilia in the pressure treated embryo measure 4 ^ as compared to the control 
cilia which are 11^. The surface cells of pressurized embryos tend to round up 
during treatment. YYhen the cilia had regenerated to a length of a few microns, 
ciliary motion was readily eyident. 



S- purpuratus 



16 



12 



8 



u 



4- 



control 




6000psi/30 min 






x/ 



40 80 120 160 

Time after deciliation (min) 

FIGURE 3. Effects of pressure on cilia regeneration in Strongylocentrotus 
embryos. Embryos at the hatched blastula stage were subjected to 6000 psi for 30 min imme- 
diately after deciliation with hypertonic seawater. Cilia regeneration of control ( ) 

and pressure treated cells ( x- -x ) are illustrated. 

A similar pattern of regeneration was found for Strongylocentrotus swimming 
blastulae. The regeneration kinetics for a representative series of embryos sub- 
jected to 6000 psi for M) min is shown in Figure 3. The regeneration delay for 
this pressure-duration treatment was 10 min in excess of treatment. This data 
suggests that the rate of regeneration was not affected by the pressure treatment. 

The effects of various magnitudes of pressure on cilia regeneration was investi- 
gated in S. f>iirf>itnitns. The duration was kept constant (30 min) but the magni- 
tude of pressure was varied systematically. In Figure 4 the cilia regeneration 
curves at three different pressure levels (2000. 4000. and 5000 psi) are shown. 
The regeneration delay was related to the applied pressure. At lower pressures the 



PRESSURE STUDIES ON CILIA 



261 



cilia were able to regenerate under compression. Short cilia \vere visible imme- 
diately following 30 min compression at 2000 and 4000 psi. At 5000 psi there 
was no regeneration during compression. As shown in Figure 4, following the 
release of pressure the regeneration profiles at the three pressure levels were com- 
parable to controls. 

DISCUSSION 

These studies show that high hydrostatic pressure is capable of profoundly 
affecting the motility of sea urchin embryos and eventually results in a loss of cilia. 
In experimentally deciliated embryos, cilia regeneration is blocked at high pres- 
sures or retarded at lower pressures for the duration of the treatment. 



18 ~ S. purpuratus 



14- 



o 
u 




2000 psi/30 min 



20 



60 100 140 

Time after deciliation (min) 



180 



E 4. Effects of pressure on regeneration in Strongylocentrotus ^iirpuratus. Imme- 
diately following deciliation by hypertonic seawater, the hatched blastulae were subjected to 
different magnitudes of pressure (2000, 4000 or 5000 psi) for a standardized duration of 30 min. 
Cilia length was plotted as a function of time after deciliation. 



Relatively few observations have been reported concerning pressure effects on 
ciliary activity in sea urchin embryos although considerable work has been done 
on other organisms such as protozoa (Kitching, 1957 and 1970; Kennedy and 
Zimmerman, 1970). In general, high pressure inhibits the activity of cilia. Tilney 
and Gibbins (1968, 1969) have reported in Arbacia embryos that pressure (6000- 
7000 psi) results in a slowing or stopping of locomotion and progressive cell dis- 
aggregation. However, they did not report any ciliary loss at these pressures for 
durations up to three hours. This is perhaps explained by the fact that their 
primary interest was the analysis of fine structure and partial ciliary loss may not 



262 p. G. YOUNG, A. D. YOUNG, AND A. M. ZIMMERMAN 

have been apparent. They reported that the microtubular elements, the basal body, 
and rootlet of the cilium were unaffected by pressures of 6000-7000 psi. Tctrahy- 
iiicna cilia, however, are quite sensitive to pressure treatment (Kennedy and 
Zimmerman, 1970). Pressures of 7500 or 10,000 psi induced a degradation of the 
central ciliary microtubules and a disorganization of longitudinal microtubules after 
durations of only 2 to 10 min. The lack of observable ultrastructural effect on sea 
urchin cilia may perhaps reflect a basic difference in ciliary structure from that 
found in Tetrahvnicini. although this is not readily apparent. The present studies 
indicate that sea urchin cilia (or associated structures) are affected by pressures 
in excess of 6000 psi. at least in the region of the base of the cilium, since pres- 
sure induces the loss of cilia as apparently intact units. The significance of the 
bulbous tips observed on detached cilia following pressure treatment is not known, 
although such tips have been observed in cilia removed by other means (Auclair and 
Siegel, 1966). In flagellae a bulbous tip can be produced by the rolling up of 
the axoneme inside the flagellar membrane ( Rosenbaum and Child. 1967). 

The mechanism by which pressure induces cilia loss is not known. On the 
basis of the present study it is difficult to speculate since even the precise level of 
amputation is in doubt. It is possible that the cytoplasmic microtubules near the 
basal body and/or the cortical plasmagel may be involved in anchoring the cilium 
since these structures have been shown to be pressure sensitive (Tilney and Gib- 
bins, 1968 and 1969; Marsland, 1970). Recently Blum (1972) has proposed 
that there is a specialized breaking point in the transitional region which lies be- 
tween the kinetosome and the ciliary shaft. 

It has been convincingly demonstrated (Auclair and Meismer. 1965 ; Auclair 
and Siegel, 1966) that initial cilia formation and regeneration do not depend on 
RNA or protein synthesis in sea urchin embryos. This implies that a considerable 
pool of the necessary proteins exists in the cells (Auclair and Siegel, 1966). 
More recent work utilizing pactamycin as a protein synthesis inhibitor indicates 
that protein synthesis may be necessary for regeneration (Child and Apter. 1969). 
This delay in regeneration may, however, be due to non-specific effects of the drug 
since earlier studies had shown that puromycin did not block regeneration although 
89% of cellular protein synthesis was inhibited (Auclair and Siegel, 1966). Inhibi- 
tion of regeneration by pressure, therefore, is probably through interference with 
assembly processes, possibly the polymerization of the microtubules. It has already 
been well established that pressure is capable of clepolymerizing and preventing 
the reformation of microtubules in the cytoplasm ( Tilney and Gibbins. 1968 and 
1969), the mitotic apparatus (Zimmerman and Marsland. 1964) and the axopodia 
of Heliozoa (Tilney ct a!., 1966). Although the intact cilium in the sea urchin 
appears to be insensitive to pressure as regards to depolymerization it is clear from 
the data that cilia formation is pressure sensitive and that growth rate can be slowed 
at lower pressures or totally inhibited at higher ones. This may be a result of a 
shift in a dynamic equilibrium between polymerized and free microtubular subunits. 
The reason for the excess delay is unknown although it probably reflects reparable 
cellular damage of some type. 

SUMMARY 
The effects of hydrostatic pressure on the cilia of sea urchin embryos (Arbacla 



PRESSURE STUDIES ON CILIA 263 

and Strongylocentrotus } were investigated. At a pressure of 10,000 psi the swim- 
ming' blastula and early gastrula embryos became less active. They lost their 
translational movement and began to rotate slowly on the bottom of the chamber ; 
in about 10 min all movement stopped and essentially all cilia had fallen from the 
embryos. At lower pressures and with longer durations the embryos were dif- 
ferentially affected and there was considerable variation in the number of cilia 
removed from individual embryos. With pressures of 6000 psi for 60 min the 
majority of the embryos lost more than 50% of their cilia. Arbacia embryos were 
more pressure-sensitive than Strongylocentrotus embryos. 

Following deciliation with hypertonic seawater, hydrostatic pressure above 
5000 psi was found to block regeneration for the duration of the pressure treatment. 
At 6000 psi and above the regeneration delay was in excess of the duration of 
pressure treatment ; the regeneration delay was directly proportional to the duration 
of treatment. At pressures lower than 5000 psi sea urchin cilia were able to 
regenerate under pressure but at a reduced rate relative to controls. Pressure 
treatment does not affect regeneration rate following decompression. 

The results arc discussed in terms of the known effects of pressure on cellular 
systems. 



LITERATURE CITED 

\i i LAIR, W., AND D. M. MEISMER, 1965. Cilia development and associated protein 

in the sea urchin embryo. Biol. Bull.. 129 : 397. 
AUCLAIR, W., AND B. W. SIEGEL, 1966. Cilia regeneration in the sea urchin embryo: evidence 

for a pool of ciliary pmidns. Science. 154: 913-915. 
BLUM, J. J., 1972. On the existence of a breaking point in cilia and flagella. /. Theor. Biol., 

' 33 : 257-203. 
CHILD, F. M., AND M. N. AFTER, 1969. Experimental inhibition of ciliogenesis and ciliary 

regeneration in Arl>acia embryos. Biol. Bull.. 137: 394-395. 
FLUGEL, H., AND C. SCHLIEPER, 1970. The effects of pressure on marine invertebrates and 

fishes. Pages 211-234 in A. M. Zimmerman, Ed., High Pressure Effects on Cellular 

Processes. Academic Press Inc., New York. 
HARVEY, E. B., 1956. The American Arbacia and Other Sea Urchins. Princeton University 

Press, Princeton, New Jersey, 298 pp. 
KENNEDY, J. R., AND A. M. ZIMMERMAN, 1970. The effects of high hydrostatic pressure on 

the microtubules of Tetrahymena pyriformis. J. Cell Biol., 47 : 568-576. 
KITCHING, J. A., 1957. Effects of high hydrostatic pressures on the activity of flagellates and 

ciliates. /. Exp, Biol.. 34 : 494-510. 
KITCHING, J. A., 1970. Some effects of high pressure on protozoa. Pages 155-177 in A. M. 

Zimmerman, Ed., High Pressure Effects on Cellular Processes. Academic Press Inc., 

New York. 
MARSLAND, D., 1950. The mechanism of cell division, temperature-pressure experiments on 

the cleaving eggs of Arlnicia punctulata. J. Cell. Comp. Physio!., 36: 205-227. 
MARSLAND, D., 1970. Pressure-temperature studies on the mechanisms of cell division. Pages 

259-312 in A. M. Zimmerman, Ed., High Pressure Effects on Cellular Processes, 

Academic Press Inc., New York. 
ROSENBAUM, J. L., AND F. M. CHILD, 1967. Flagellar regeneration in protozoan flagellates. 

/. Cell Biol.. 34: 345-364. 

ROSENBAUM, J. L., J. E. MOULDER AND D. L. RINGO, 1969. Flagellar elongation and shorten- 
ing in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis 

and assembly of flagellar proteins. /. Cell. Bio!.. 41 : 600-619. 



264 I'. G. YOUNG, A. D. YOUXG, AND A. M. ZIMMERMAN 

TILNEY, L. G., AND J. R. GiBBiNS, 1968. Differential effects of antimitotic agents on the 
stability and behaviour of cytoplasmic and ciliary microtubules. Protopla-sina, 65: 
167-179. 

TILNEY, L. G., AND J. R. GIBBINS, 1969. Microtubules in the formation and development of 
the primary mesenchyme in Arbacia punctiilata. II. An experimental analysis of their 
role in development and maintenance of cell shape. /. Cell. BioL, 41 : 227-250. 

TILNEY, L. G., Y. HIRAMOTO AND D. MARSLAND, 1966. Studies on the microtubules in 
Heliozoa. III. A pressure analysis of the role of these structures in the formation 
and maintenance of the axopodia of Actinosphaerium nucleofilum (Barrett). /. Cell. 
Biol.,29: 77-95. 

ZIMMERMAN, A. M., Ed., 1 ( '70. /-////// Pressure Effects mi Cellular Processes. Academic 
Press Inc., New York, 324 pp. * 

ZIMMERMAN, A. M., AND D. MARSLAND, 1964. Cell division: Effects of pressure on the mitotic 
mechanisms of marine eggs (Arbacia pttuctulata) . /:.r/>. Cell. Res., 35: 293-302. 



Vol. 143, No. 2 October, 1972 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



CHEMOTACTIC AND GROWTH RESPONSES OF MARINE 
BACTERIA TO ALGAL EXTRACELLULAR PRODUCTS 

WAYNE BELL AND RALPH MITCHELL 

Department of Biology, Middlebury College, Middlebury, Vermont 05753; Laboratory of 

Applied Microbiology, Division of Engineering and Applied Physics, Harvard 

University, Cambridge, Massachusetts 02138; and the Marine Biological 

Laboratory, Woods Hole, Massachusetts 02543 

It has been known for many years that filtrates from axenic algal cultures may 
be enriched with organic compounds. These materials, including simple amino 
acids and peptides, sugars, polyalcohols, and occasionally vitamins, enzymes, and 
toxins, are usually lumped under the term "extracellular products" (Fogg, 1966). 
Studies using natural populations of phytoplankton have shown that extracellular 
products are not mere laboratory artifacts, and that, depending upon environmental 
conditions, they account for 1-20% of the total photoassimilated carbon (Helle- 
bust, 1965; Nalewajko, 1966; Samuel, Shad and Fogg, 1971; Thomas, 1971). 

The potential significance of extracellular organic material in marine food chains 
is extremely interesting. Many authors (Fogg, 1966; Brock, 1966; Alexander, 
1971 ; Whittaker and Feeney, 1971) have suggested that these products may play 
an important role in marine food chains, especially as potential nutrients for bac- 
teria. However, to our knowledge, there is no direct evidence that this is so 
although the ability of bacteria to grow in algal cultures (Vela and Guerra, 1966; 
Berland, Bianchi and Maestrini, 1969) might be interpreted to support such 
conclusions. 

If, in fact, algal extracellular products are important contributors to bacterial 
food chains, it would seem possible to construct an aquatic counterpart of the well- 
known "rhizosphere" of terrestrial ecosystems (Rovira, 1965). A zone may exist, 
extending outward from an algal cell or colony for an undefined distance, in which 
bacterial growth is stimulated by extracellular products of the alga. For purposes 
of discussion in this paper, we will term this region the "phycosphere." 

Motile bacteria commonly exhibit chemotaxis to concentration gradients of or- 
ganic material (Weibull, 1960; Adler, 1969). The ecology of chemotaxis by 
organotrophic bacteria has not been well studied, but highly species-specific re- 
sponses to certain carbohydrates, amino acids, and nucleotide bases have been ob- 
served (Fogel, Chet and Mitchell, 1971), and certain predatory microorganisms 
have been shown to be chemotactic to their prey (Chet, Fogel and Mitchell, 1971). 

265 

Copyright 1972, by the Marine Biological Laboratory 
Library of Congress Card No. A38-518 



266 W. BELL AND R. MITCHELL 

It is possible that chemotaxis may also be of importance in the establishment of a 
phycosphere microflora. 

In the current studies, we have investigated the validity of the phycosphere 
concept in a number of ways. We have especially been concerned with the existence 
and importance of the phycosphere effect during various stages of algal cell growth 
and death, and the relationship of bacterial chemotaxis to the establishment and 
maintenance of a phycosphere microflora. 

.\ I ATERIALS AND METHODS 

Axenic cultures of marine algae were kindly supplied by R. R. L. Guillard, 
Woods Hole Oceanographic Institution. The following were used in the studies 
reported here: Skelctonema costatum (clone SKEL), Cyclotella nana (clone 3H), 
Dunaliella tertiolecta (clone DUN), Isochrysis galbana (clone ISO). 

The algae were maintained on culture medium F/2A, a slight modification of 
sea water enrichment medium F/2 (Guilliard and Ryther, 1962), containing 1000 
(J.M. NaNO 3 and 50 /*M K 2 HPO 4 per liter. All cultures were grown in 50-ml 
batches in 125-ml Erlenmeyer flasks at 16 C with 900 foot-candles fluorescent 
illumination supplemented by four 25 w incandescent bulbs ; lights were programmed 
to an 18-hr-on, 6-hr-off cycle. Cell counts were made using a brightline hemocytom- 
eter (American Optical Co., #1492). Cultures were routinely transferred every 
10 days. 

Algal culture filtrates were obtained by centrifuging aliquots 5 min at 1000 rpm 
in a table-top clinical centrifuge (International Equipment Co., model CL), followed 
by sterile filtration through 0.45-/mi membrane filters (Millipore HA) which had 
been pre-washed with a total volume of 10 ml synthetic sea water. Microscopic 
examination of cells following centrifugation and filtration failed to reveal any de- 
tectable cell damage not already in the cultures being sampled. In order to cancel 
the effects of any organic material introduced into the filtrates from the Millipore 
filters, F/2A, synthetic sea water, and natural sea water were similarly filtered 
when used as controls in chemotaxis experiments. All filtrates were stored frozen 
in screw-capped vials until just before use. 

Bacteria were cultured on medium I SOL having the following ingredients per 
liter: 5.0 g peptone (Difco), 1.0 g yeast extract (Difco), 0.001 g K 2 HPO t filtered 
or synthetic sea water to volume. When used for plating, this medium was 
solidified with 1.5% agar (Difco). 

The synthetic sea water medium (SSW) used in these experiments had the fol- 
lowing composition per liter: 1.13 g CaCl 2 , 7.0 g MgSO 4 , 5.3 g MgQ 2 -6H 2 O, 
0.72 g KC1, 25 g NaCl, distilled water to volume. 

The composition of J SW was chosen to approximate the composition of natural 
sea water in terms of the most abundant salts. It also satisfies the major ionic 
requirements of most marine bacteria (MacLeod, 1965). The general suitability of 
this synthetic sea water is reflected in the fact that all bacterial isolates obtained dur- 
ing these studies could readily be grown on ISOL in which SSW had been substi- 
tuted for natural sea water. Of course, all bacterial media are selective in one way 
or another, and the media reported here are no exceptions. Problems introduced 
by our "selective" media were not evaluated, but they are believed to be minor. 
Bacterial isolates were obtained from enrichments of aseptically-obtained sea 



RESPONSES OF BACTERIA TO ALGAE 



267 



CO 



UJ 

o 



C3 



U_ 
O 

d 



q 6 



-* SKELETONEMA ALONE 

-o SKELETONEMA + ISOLATE 

7697 




_L 



12 18 

CULTURE AGE (DAYS) 



24 



FIGURE 1. Growth of Skeletonema costatum in batch culture in the presence and absence 
of the bacterial isolate 7697. No effect of the bacterium on algal growth was evident in these 
experiments. 



water samples taken from Vineyard Sound near Woods Hole, Massachusetts. The 
enrichment, begun within 2 hrs after obtaining a sample, consisted of 5 ml of 
sample + 50 ml of ISOL in 125-ml Erlenmeyer flasks. These were incubated with 
shaking at room temperature (approximately 27 C) for 18-36 hr before use. 
These techniques essentially selected for those bacteria able to grow rapidly on a 
rather rich complex medium. There was no particular selection for specific nu- 
tritional types other than for bacteria able to grow aerobically. A more specific 
technique used to obtain bacteria responding to algal culture filtrates is reported 
later in this paper. Overnight enrichments always contained a large number of 
highly motile bacteria. 

Chemotactic assays of bacteria, whether obtained directly from mixed enrich- 
ments or from pure isolates, were complicated by a variety of behavioral nuances, 
including the rapid settling of some bacteria onto surfaces and general loss of 
response upon repeated subculturing. These problems were not completely over- 
come, but the following procedure yielded the most reliable bacterial preparations: 
50-ml cultures were grown on ISOL overnight at room temperature with shaking: 
3-6 hr before an experiment, a 2-ml aliquot was inoculated into a second SO ml of 
ISOL, After the culture became turbid, the cells were harvested by centrifugation 
10 min in a clinical table-top centrifuge (International Equipment Co., model CL) 



268 



W. BELL AND R. MITCHELL 



at 2800 rpni; although the supernatant remained slightly turbid, an appreciable 
pellet was always obtained. The supernatant was drawn off and the cells resus- 
pended in 5 ml sterile SSW. After a second centrifugation, the cells were re- 
suspended again in sterile SSW to a concentration of 10 G -10 7 per ml. Microscopic 
examination of such preparations showed that 10-90% of the bacteria were motile; 
only preparations having a high degree of motility were utilized in experiments. 

Bacterial chemotaxis was assayed using the method of Adler (1969). Agar 
plugs were used to close the 1 p.1 capillaries. Chemotaxis experiments were run 
at room temperature lor 15 to 45 min. During this time bacterial motility and 
behavior was monitored visually in selected preparations and controls with a phase 
contrast microscope. At the end of an experiment the contents of each capillary 
tube were diluted into 10 ml of sterile SSW and bacterial counts made by plating 
aliquots on solidified I SOL. Chemotaxis experiments were run in duplicate 
or triplicate. 



* SKELOTONEMA + 7697 
CYCLOTELLA + 7697 
F/2A MEDIUM + 7697 




6 12 18 

CULTURE AGE (DAYS) 

FIGURE 2. Growth of the bacterial isolate 7697 in the presence and absence of two algae, 
Skeletonema costatum and Cycloiclla nana. The Skcletonema culture is the same one depicted 
in Figure 1. Bacteria were inoculated into these cultures at 0.5 X 10* per nil. 



RESPONSES OF BACTERIA TO ALGAE 



269 



RESULTS 

In the first series of experiments, we examined the growth of mixtures of 
algae and bacteria. These experiments were designed to test for production of 
either anti-bacterial toxins or bacterial stimulants by the algae. Pure cultures of 
motile bacterial isolates were washed free of growth medium by centrifugation 
and resuspension in sterile SSW, then inoculated into freshly transferred axenic 
algal cultures. Controls consisted of separate algal and bacterial cultures in F/2A 
medium inoculated at the same time and incubated under the same conditions. The 
only major carbon source for the bacteria consisted of extracellular material 
produced by the algae. 

Typical results from these experiments are shown in Figures 1 and 2. The 
two bacterial isolates tested had no discernible effect on algal growth. However, 



SKELETONEMA FILTRATE 
CYCLOTELLA FILTRATE 




12 18 24 

AGE OF ALGAL CULTURE (DAYS) 



30 



FIGURE 3. Chemotactic response of bacterial isolate 7697 to filtrates from algal cultures of 
increasing age. Values on the ordinate are given as the ratio of the number of cells in capillary 
tubes containing algal culture filtrate to the number in tubes containing F/2A medium. 
Experiments lasted 45 min. at room temperature. 



viable cell counts showed that the bacteria were strongly affected by the presence 
of algal cells. There was typically an initial burst of bacterial growth during the 
first week of culture. This was followed by a period of either no increase or a sig- 
nificant decrease in viable cell count, approximately coinciding with the transition 
period between logarithmic and stationary algal growth. Invariably a marked 
increase in viable bacterial cells was observed as the cultures aged further. 

When compared with the experimental flasks, bacterial growth in the controls 
was insignificant. The bacterial concentration was always several orders of mag- 
nitude less. A slight increase in viable cells during the first week probably repre- 
sents growth on the organic material present in the natural sea w r ater base of F/2A 



270 



W. BELL AND R. MITCHELL 



medium ; the gradual increase with age can be accounted for by evaporation of the 
medium in the flasks. 

It is evident that the bacteria tested are able to coexist in culture with these 
algae. The data suggest that most of these bacteria depend on degradation products 
of the algal cells. A small residual population of bacteria existed in a viable state 
throughout the 30-day period tested, even in control flasks. 

In order to study the possible ecological role of these materials further, an assay 
utilizing bacterial chemotaxis was developed and employed in a series of experi- 
ments with intentions similar to those reported above i.e., the determination of the 
effect of algal culture age on bacterial response. Controls for these experiments 
consisted of capillary tubes containing filtered F/2A of the same age as the algal 
culture being tested. 

The results of one such experiment are shown in Figure 3. The bacterium was 
isolate 7697, one of those used in the previous growth experiments. The chemo- 
taxis pattern obtained is typical and can profitably be compared with the growth 
data of Figure 2. Filtrates from young algal cultures did not attract the bacterium. 



300 r- 



ISOLATE 8716 
ISOLATE 8715 
ISOLATE 8714 




20 30 

MINUTES 



40 



50 



FIGURE 4. Attraction of bacteria from enriched Vineyard Sound water to filtrate from 
30-day Skelctonema culture. These bacteria, from the same water sample, could be readily 
differentiated on plates on the basis of colony morphology and color. No other bacteria 
appeared in significant numbers in this experiment. 



RESPONSES OF BACTERIA TO ALGAE 



271 



but chemotaxis increased markedly when filtrates from older cultures were used 
as attractants. 

Taken together, the data presented in Figures 1-3 strongly suggest the stimu- 
lation of a "phycosphere effect" by the algae. However, the data obtained indicate 
that algal excretions are important only after algae have ceased rapid growth and 
commenced decomposition. 

A series of experiments were initiated to examine the ability of algal culture 
filtrates to select for specific bacteria from a mixed population. This process is 
critical in the construction of a phycosphere effect mediated by algal extracellular 
products. We utilized filtrates from 30-day algal cultures as attractants. These 
were selected because of their maximum attractiveness to bacteria in previous ex- 
periments. Adequate controls were difficult to construct, but Millipore filtered algal 
medium F/2A of the same age was used as the best compromise. Because bac- 
terial populations in natural sea water are too low to assay quantitatively by this 

TABLE I 

Attraction of bacterial isolates to filtrates from 30-day algal cultures and to a peptone solution. 

Data are expressed as for the ordinate in Figure J; 30-day old uninoculated F/2A 

medium was used in control tubes. Bacterial response is statistically 

significant (P = 0.05) if the ratio is 2.0 or greater 



Algal culture filtrates 


Bacterial clone 


8712 


8714 


8715 


8716 


No. of bacteria attracted/No, attracted to control medium 


Skeletonema 


4.2 


3.2 


2.9 


65 


Cyclotella 
Dunaliella 


3.4 
3.3 


5.2 
3.4 


2.1 


95 

122 


Isochrysis 
0.5% peptone 


0.3 
7.9 


3.1 

3.3 


0.8 
6.7 


51 
46 



technique, overnight enrichments of whole water samples were tested. No attempt 
was made to isolate "dominant" bacteria from the water samples. This is justifiable, 
since any true phycosphere would also be an enrichment bearing little relation to 
the dominant bacteria outside the zone of influence (Rovira, 1965). 

Figure 4 shows the results of one such experiment using a mixed bacterial en- 
richment from Vineyard Sound. The three bacterial types indicated could readily 
be distinguished on the basis of colony appearance on the counting plates. Other 
bacteria may have been present but were not seen at the dilutions counted. These 
data indicate that algal culture filtrates are indeed capable of eliciting chemotactic 
responses from a non-specific enrichment, and presumably from indigenous bacteria 
in the water column, with the degree of response differing between bacterial types. 
Such observations can be exploited in the laboratory for the purpose of obtaining 
bacteria that respond to specific algal species. Because these experiments utilized 
a mixed bacterial system, however, caution must be used in labelling bacteria as 
"strong" or "weak" in their response, as the presence of other bacteria may lead 
to undefinable interactions. 



272 



W. BELL AND R. MITCHELL 



During the visual monitoring of this particular experiment, variations in bac- 
terial behavior could be readily discerned and later correlated with the specific iso- 
lates obtained. The Spirillum, isolated as clone 8716, entered the capillary tube 
extremely rapidly and within 15 min could be found along the entire length. Such 
behavior is characteristic of spirilla and is often utilized in their isolation (Veldkamp, 
1970). The small pseudomonad, isolated as clone 8715, entered less rapidly but by 
the end of the experiment it had formed a band of high concentration just inside 
the mouth of the tube. Isolate 8714 was at too low a concentration in the tube to 
be studied visually. 

After their isolation, bacterial strains were tested separately for their ability 
to respond to 30-day algal culture filtrates and compared with their response to 
0.5% peptone, a rather rich organic attractant (Table I). In general, the isolates 
responded to the culture filtrates as well as, or better than, peptone, with the 
exception of Isoc/irysis filtrate. The best response was shown by isolate 8716, 
paralleling its behavior in the mixed enrichment. Despite the possibility of inter- 
specific bacterial interactions in the mixed enrchments, the data of Table I confirm 
that the bacterial responses are independent of other bacteria. 

From the above data it can be concluded that specific bacteria may be selected 
from a non-specific mixture by algal extracellular products. This selection would be 
mediated by the chemotactic responses of the bacteria. Subsequent experiments 
were designed to evaluate this possibility to see if the laboratory studies could be 
extrapolated into natural systems. 



TABLE 1 1 

ey of bacterial chemotactic responses to chemicals identified in filtrates from marine algal 
cultures. Data expressed as for ordinate in Figure 3, with SSW serving as a control 
The response is significant (P = 0.05) if the ratio is greater than 2.0. All 
chemicals were tested at a concentration of 10~ 2 M in SSW 





Bacterial clone 


Attractant 


8712 


8714 


8715 


8716 




No. of bacteria attracted/no, attracted to control mediu 


Aniiao acids: 










alanine 


4.3 


1.3 


7.2 


24 


valine 


5.3 


0.7 


5.3 


24 


proline 


1.7 


1.3 


4.6 


9.5 


lysine 


2.1 


0.4 


3.4 


146 


arginine 


8.9 


1.4 


5.6 


87 


methionine 


8.6 


2.5 


4.1 





glutamic acid 


0.2 


0.0 


0.1 


0.0 


aspartic acid 


0.0 


0.0 


0.0 


0.0 


Polyalcohols: 










mannitol 


0.8 


1.3 


1.8 


43 


glycerol 





1.5 


1.5 


1.0 


Sugars: 




^ 

M 






glucose 


1.1 


1.8 


1.0 


1.7 


sucrose 


1.3 


4.9 


2.2 


39 



RESPONSES OF BACTERIA TO ALGAE 273 

The construction of adequate control experiments is difficult because of the 
labile nature of algal culture media. These can change their properties with age 
whether algae are present or not (Provasoli, McLaughlin and Droop, 1957). We 
did not detect any significant change in any of our media during thirty days of 
storage. F/2A was therefore used routinely when testing for chemotaxis to algal 
culture filtrates. However, SSW, expected to have a lower background concen- 
tration of organic material, was employed when testing for chemotaxis to specific 
chemicals. 

Hellebust (1965) reported on the nature of extracellular products from many 
marine phytoplankters, including those algae studied here. These compounds 
included simple amino acids, sugars, and polyalcohols ; extracellular peptides and 
polysaccharicles were also implicated by the increase in amino acids and mono- 
saccharides after acid hydrolysis of culture filtrates. 

If extracellular products are to be implicated as bacterial attractants or related 
in any other way to the establishment of a phycosphere microflora, the compounds 
identified by Hellebust (1965) would be expected to be among the active components. 

Several of the fresh bacterial isolates shown to be chemotactic to algal culture 
filtrates were tested for their chemotactic response to Hellebust's (1965) extra- 
cellular products. The results (Table II) show that the amino acids elicited the 
best responses. Curiously, the common metabolite glucose was not a good at- 
tractant, though most bacteria responded to sucrose. Glycerol and mannitol, 
identified in extracellular products by Hellebust (1965), were generally not atrac- 
tive. The response to glutamic and aspartic acids was low, due to inhibition of bac- 
terial motility around the mouth of the capillary tube. When these compounds 
were tested dissolved in sea water, there was no chemotactic response although 
motility was not inhibited. 

The absolute concentrations of specific compounds among the extracellular 
products of marine algal cultures is technically difficult to determine because of 
problems associated with desalting the medium prior to concentrating the organic 
material. Some preliminary experiments in this laboratory, studying the extra- 
cellular production by the marine alga Chlorella sp. (Woods Hole clone 580) indi- 
cate concentrations of 10~ 8 -10~ 6 M for amino acids and sugars found in the filtrates 
of log phase cultures. 

Experiments to determine the threshold for bacterial chemotactic response were 
constructed to compare with such information. These experiments consisted of 
chemotaxis assays of single bacterial preparations using increasing tenfold dilutions 
of selected organic compounds in SSW. In almost all cases, the values were found 
to lie between attractant concentrations of 1O 6 -10~ 5 M. This range agrees well 
with threshold concentrations found for chemotactic responses of Escherichia coll 
to monosaccharides as reported by Adler (1969). Considering the very low con- 
centrations of organic material found in algal culture material and likely to be 
found in natural waters, these thresholds are surprisingly high. 

DISCUSSION 

The construction of a theory to account for the development of a phycosphere 
is dependent on two criteria. There must be a source of enrichment for the mi- 
crobial population in proximity to the algae. The microflora must respond to the 



274 W. BELL AND R. MITCHELL 

algal products by being attracted and/or growing in this region. The data 
presented will be discussed from the point of view of these criteria. 

The chemical nature of algal extracellular products renders them likely sources 
of microbial nutrients. As they may constitute a significant portion of primary 
production, such compounds are indeed of potential significance in microbial food 
chains. There is considerable confusion in the literature, however, as to the source 
of these compounds. Short-term experiments such as the ones of Fogg, Nalewajko 
and Watt (1965) and Watt and Fogg (1966) strongly suggest that the materials 
may be released as products of cell metabolism, a process sometimes termed "ex- 
cretion." On the other hand, long-term experiments lasting several days such as 
those of Marker (1965) have shown that the increase in soluble organic carbon in 
algal culture filtrates might readily be attributed to cell lysis, on the order of 1 
in 100-1000 cells daily. Accurate determination of cell lysis by counting techniques 
has so far not proved technically feasible. 

This difficulty in determining the actual source of extracellular organic material 
under natural conditions complicates ecological interpretation of the effect of this 
material on bacterial populations. In our study (Figs. 1 and 2) we were able to 
demonstrate that bacteria were indeed able to grow in algal cultures with no addi- 
tional carbon source an observation known to all workers who routinely isolate 
algal cultures and by no means a novel one (Berland, Bianchi and Maestrini, 
1969). 

The data contain two additional important observations, however. There was 
no discernable predation of bacteria on the algae. This reinforces the conclusion 
that the material on which the bacteria were growing was indeed extracellular. In 
addition, bacterial growth was maximal during the declining stage of the algal 
growth curve, when algal cell lysis was evident. It was often possible to observe 
the presence of bacterial aggregates around clumps of lysed algal cells. 

The observations shown in Figure 2 also include an increase in bacterial con- 
centration during the early stage of algal growth. The source of organic material 
for this increase has not been determined, although it probably was extracellular. 
It appears, however, that algal extracellular products may have the greatest im- 
pact on the bacterial community only during the latter stages of a phytoplankton 
bloom, when algal cell lysis is highest. 

The second criterion for the establishment of a phycosphere, bacterial re- 
sponse, was studied in more depth. The data show conclusively that bacteria are 
capable of growing in algal cultures. The behavioral response, in this case chemo- 
taxis, was studied from the belief that if a phycosphere were ecologically sig- 
nificant, motile bacteria might be attracted to this region before commencing growth 
on the organic material. 

Our data indicate that marine bacteria are chemotactic to algal culture filtrates. 
This response was invariably highest to filtrates from old algal cultures (Fig. 3), 
implying that the release of extracellular carbon is most important ecologically 
during the later stages of a plankton bloom. There was no significant chemotactic 
response to filtrates from younger cultures, even though such cultures supported 
bacterial growth. 

The threshold concentrations of some " of the compounds eliciting bacterial 
chemotaxis were found to lie generally in the range 10~ 5 -10" 4 M. Nearshore waters 



RESPONSES OF BACTERIA TO ALGAE 

and estuaries usually average 10" 6 -10" D M for carbohydrate and 10~ 8 -10~ 7 M for 
specific amino acids (Wagner, 1969). Bacteria were not attracted to natural sea 
water (compared against SSW) in our experiments. Utilizing bottles incubated 
in situ in freshwater lakes, Fogg and Watt (1965) found that Chlorella pyrenoidosa 
produced a maximum concentration of extracellular glycollic acid of 1.5 mg/1, or 
about lO r ' M. In our own laboratory, the concentrations of extracellular amino 
acids and sugars in algal cultures are at best an order of magnitude less during 
logarithmic growth. In both cases, such experiments have utilized closed sys- 
tems in which the extra cellular products could accumulate unnaturally. Thus, 
relative to the general concentrations of organics found in nature, the thresholds 
for bacterial chemotaxis are very high. 

These data do not support the second criterion for a phycosphere as far as 
rapidly growing planktonic algae are concerned. No bacterial response to young 
algal culture filtrates was observed. While the possibility of bacterial inhibition 
cannot be eliminated completely, observations of bacterial behavior during such 
experiments failed to reveal any noticeable inhibition of bacterial motility or general 
activity. The interpretation most consistent with these data is that the concen- 
trations of extracellular compounds in filtrates from young algal cultures simply 
were not above the required thresholds for bacterial attraction. In the filtrates 
from older algal cultures, it would appear that the second criterion for the creation 
of a phycosphere is met. at least in terms of bactrial chemotactic response to these 
filtrates. 

Bacterial chemotaxis probably serves to keep a bacterial cell near a source of 
organic material once it has arrived there by chance. A decomposing algal cell 
thus serves as a bacterial sink. The shock reaction behavior does not aid a bac- 
terium in locating such a sink, but keeps it there once the bacterium gets close 
enough to respond to the chemical gradient. This effect may be further enhanced 
by the tendency of motile bacteria to settle onto nearby surfaces rapidly after they 
begin exhibiting shock reactions in response to a supra-threshold concentration of 
chemicals. This kind of behavior is very similar to that observed in photosynthetic 
bacteria responding to a restricted zone of illumination (Pfennig, 1967). Such 
behavior would help explain the ability of marine bacteria to exist in what is 
otherwise in terms of dissolved organic material a nutritionally poor environ- 
ment (Jannasch, 1967). 

The notion that algal extracellular products must be highly significant in 
bacterial food chains is far too general to be of much predictive use. The data 
presented here indicate that a more narrow definition of "extracellular products" 
is in order, as the bacterial growth and chemotactic response varies greatly 
with the age of algal cultures and these are, in fact, greatest when algal cells 
are lysing in old cultures. The role of extracellular compounds released by 
rapidly growing algae remains to be evaluated, but the general statement that 
they have a great significance in microbial ecology is totally unwarranted 
unless qualified by considering highly specific classes of compounds such as anti- 
biotics, vitamins, toxins, etc. 

We have found that the term "phycosphere" can be a useful one in discussing 
and evaluating algal-bacterial interrelationships. The phycosphere effect would 
be expected to be greatest during algal bloom decomposition. This effect is medi- 



276 w. BELL AND R. MITCHELL 

ated in part by bacterial chemotaxis to organic material released by the lysing 
algal cells, which serves to keep bacteria in proximity to the cells until most of 
the available organic material has been utilized. It would appear that the phyco- 
sphere is a region of interactions that have only begun to be evaluated. 



We thank Miss J. Lang for excellent technical assistance during portions of 
this work. The research was supported in part by contract #NOOO 14-6 7-0298- 
0026 between Harvard University and the Office of Naval Research. 

SUMMARY 

1. The possibility that planktonic algae possess a "phycosphere," a zone sur- 
rounding them created by the production of extracellular products which may serve 
as bacterial nutrients, is examined. 

2. Bacterial growth in algal cultures to which no additional organic material is 
added is greatest only as the cultures age and algal cell lysis becomes obvious. 

3. Marine bacterial isolates are chemotactic to nitrates from algal cultures, but 
the response is significant only to filtrates from old cultures, again where cell 
lysis is evident. 

4. Specific compounds known to occur as algal extracellular products attract 
bacteria, but the threshold concentrations for attraction are unexpectedly high 
when compared with the generally very low concentrations of organic compounds 
in natural sea water. 

5. The validity of the phycosphere concept and its potential importance to 
marine microorganisms is discussed. 

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RESPONSES OF BACTERIA TO ALGAE 277 

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SAMUEL, S., N. M. SHAH AND G. E. FOGG, 1971. Liberation of extracellular products of photo- 
synthesis by tropical phytoplankton. /. Mar. Biol. Ass. U. K., 51 : 793-798. 
THOMAS, J. P., 1971. Release of dissolved organic matter from natural populations of marine 

phytoplankton. Mar. Biol., 11 : 311-323. 
VELA, G. R., AND C. H. GUERRA, 1966. On the nature of mixed culture of Chlorclla pyrcnoidosa 

TX71105 and various bacteria. /. Gen. Microblol., 42 : 123-131. 
YKLDKAMP, H., 1970. Enrichment cultures of prokaryotic organisms. Pages 305-361 in J. R. 

Norris and D. W. Ribbons, Eds., Methods in Microbiology, 3a. New York, Academic 

Press. 
WAGNER, F. S., JR., 1969. Composition of the dissolved organic compounds in seawater : a 

review. Contrib. Mar. Sci., 14: 115-153. 
WATT, W. D., AND G. E. FOGG, 1966. The kinetics of extracellular glycollate production by 

Chlorella pyrenoidosa. J. Exp. Bot., 17: 117-134. 
WEILBULL, C., 1960. Movement. Pages 153-205 in I. C. Gunsalus and R. Y. Stanier, Eds., 

The Bacteria, I. New York, Academic Press. 
WHITTAKER, R. H., AND P. P. FEENY, 1971. Allelochemics : chemical interactions between 

species. Science, 171 : 757-770. 



Reference: Biol. Bull., 143: 278-295. (October, 1972) 



RESPONSES OF CHAETOPTERUS VARIOPEDATUS TO OSMOTIC 

STRESS, WITH A DISCUSSION OF THE MECHANISM 

OF ISOOSMOTIC VOLUME-REGULATION 

STEPHEN C. BROWN, JOHN B. BDZIL AND HARRY L. FRISCH 

Department of Biological Sciences and Department of Chemistry, 
Stole University of New York, Albany, New York 12222 

Detailed studies on salt and water balance in polychaetes have been primarily 
directed toward free-living intertidal and estuarine species (see Olgesby, 1969a 
and 1969b, for extensive reviews). Less information is available on sedentary 
tube-dwelling worms particularly those which appear to be stenohaline. One 
such species, Chaetopterus variopedatus, occurs widely along coastal areas in the 
intertidal to subtidal zones. Although it is usually found in regions of stable 
high salinity, it has been reported from areas where the salt concentration reaches 
as low as 20% (Gosner, 1971). In addition, Chaetopterus is known to occur 
intertidally in areas of heavy seasonal rainfall which may be presumed to produce 
some dilution of the interstitial seawater. It has been pointed out, however, 
that the actual salinities immediately surrounding burrowing or tube-dwelling 
organisms may be considerably different than a cursory sampling of the surround- 
ing water would suggest (Gunter, 1961; Oglesby, 1969b). It is of interest in 
this connection that Garrey (1905) could find no evidence that Chaetopterus 
could regulate its salt or water content when subjected to salinities below that of 
approximately 31% . The present investigation was undertaken, therefore, to 
examine in greater detail the responses of Chaetopterus to osmotic stress. 

MATERIALS AND METHODS 

Source and maintenance of animals 

Specimens of Chaetopterus were obtained from Pacific Bio-Marine Supply 
Co., Venice, California. In the laboratory, the animals (within their natural 
tubes) were maintained at 16 C in aerated recirculating aquaria containing 50 
gallons of artificial seawater ("Instant Ocean," Aquarium Systems, Inc., Cleve- 
land, Ohio). Worms were fed ad libitum daily with "Fryfare" extra fine fish 
food (Wardley Products Company, Inc., Long Island City, New York). Animals 
were acclimated for a minimum of 7 days under these conditions before any ex- 
periments were performed. 

Preparation of test solutions 

Hypoosmotic solutions were prepared by diluting the artificial seawater in 
which the worms had been maintained with an appropriate amount of distilled 
water. 

278 



OSMOTIC RESPONSES OF CHAETOPTERUS 279 

Weight determinations 

For the determination of wet weights, the animals were blotted dry with 
absorbent paper towels and weighed in air (at 16 C) to the nearest milligram 
on a torsion balance (The Torsion Balance Co., Clifton, New Jersey). 

Collection of co domic fluid samples 

Prior to removal of coelomic fluid, the animals were thoroughly blotted with 
absorbent paper towels. With the aid of a binocular dissecting microscope, the 
tip of a 200 /JL\ capillary tube, drawn out to a fine point, was inserted through the 
integument into the large coelomic cavity of one (or more) of the "fan" segments. 
Fluid was then drawn into the capillary with the aid of a mouth suction-tube. 
Depending on the size of the animal, from 50 to 150 n\ of fluid could be obtained 
from a single segment in this fashion. A final sample from each worm (in excess 
of 125 fj.\) was obtained by pooling the coelomic fluid from the three fan segments, 
as needed. The capillary tubes were flame-sealed and stored at --20 C. Since 
coelomic fluid from these segments was virtually devoid of cells, the samples 
were not centrifuged prior to making determinations. 

Determination of osmotic pressure, Na + and Cl~ 

Osmotic pressure was measured with an Advanced High Precision freezing- 
point osmometer (Advanced Instruments, Inc., Newton Highlands, Massa- 
chusetts) in the following manner. Duplicate 50 n\ samples of coelomic fluid 
were diluted to 200 n\ with glass-distilled water and placed in sample holders 
(0.2 ml) for determination. The final results were calculated by comparison to a 
standard curve made from solutions of known osmolarity (standard solutions from 
Advanced Instruments, Inc.) with correction for electrolyte activity change 
caused by dilution. 

For sodium determinations, duplicate 5 /*! samples of coelomic fluid were 
diluted to 5.00 ml with glass-distilled water. Flame photometry was carried out 
using a Coleman Jr. II spectrophotometer with flame attachment, and the results 
compared to a standard curve made from solutions of known sodium concentration. 

For chloride determinations, duplicate 5 fj,\ samples of coelomic fluid were 
diluted to 50 /JL\ with glass-distilled water. Titrimetric determinations were made 
using an Oxford automatic titrator (Oxford Laboratories, San Mateo, California) 
with acid mercuric nitrate in the presence of s-diphenylcarbazone as an endpoint 
indicator. Results were compared to standard solutions of known chloride 
concentration. 

RESULTS 
Long-term adaptation to hypoosmotic seawater 

To examine the response to long-term, gradual lowering of salinity in the ex- 
ternal medium, 50 worms (in their natural tubes) were allowed to acclimate for 
one week in artificial seawater of 1090 milliosmoles concentration. The health 
of the worms was estimated by cutting off one of the tips of the "U 1 '-shaped tubes 
and observing whether or not the tip was repaired. Rapid (overnight) repair of 
its tube house was taken as a sign that a worm was in good condition. Five 



280 



BROWN, BDZIL, AND FRISCH 



worms were removed from their tubes and coelomic fluid and medium samples 
taken. The salinity in the acclimation aquarium was then gradually lowered 
at a rate of approximately 3% per day by addition of calculated amounts (volu- 
metric) of distilled water. At varying intervals, groups of six worms in their 
tubes were transferred to other aquaria and kept for seven days at the lowered 
salinity attained. All worms were fed daily. At the end of the week period, 
worms were removed from their tubes, examined, and coelomic fluid and medium 
samples taken. The data for total osmolarity, sodium, and chloride concentra- 
tions are shown in Figures 1-3. 

Over the range studied, it appears that the coelomic fluid of Chaetopterus 
conforms to the external medium with respect to total osmolarity and sodium and 



I 

a 



\\00 



\ooo 



900 



800 



700 



600 



600 700 800 900 

Medium, milliosmoles 



1000 



1100 



FIGURE 1. The osmotic concentration of Chaetopterus coelomic fluid in relation 
to that of the external medium, all points shown. 

chloride concentration. There is no indication of ionic or osmotic regulation for 
the parameters examined. Only a single animal (out of 10) survived the slow 
dilution and week-long maintenance at 630 milliosmoles (i.e., 57.8% of the initial 
value). It would appear then, that approximately 55-60% seawater is the lower 
limit which Chaetopterus can tolerate for moderately extended periods of time. 
Survivorship and/or regulation was not examined in hyperosmotic solutions 
exceeding ca. 1100 milliosmoles. 

Volume changes during osmotic stress 

To examine the effects of osmotic stress on water and salt movements, worms 
were carefully removed from their tubes and placed individually in 4-inch finger 



OSMOTIC RESPONSES OF CHAETOPTERUS 



281 



500 - 



450 - 



I 



400 - 



350 - 



300- 



250 - 




250 300 350 400 

Medium, mM No* 



450 



500 



FIGURE 2. The sodium concentration of Chaetopterus coelomic fluid 
in relation to that of the external medium, all points shown. 



600 



550 



500 



450 



,0 



400 



350 



350 400 450 500 

Medium, mM Cl~ 



550 



600 



I-'U.URE 3. The chloride concentration of Chaetopterus coelomic fluid 
in relation to that of the external medium, all points shown. 



282 



BROWN, BDZIL, AND FRISCH 



bowls filled with 200 ml of artificial seawater. They were then carefully examined , 
and any appearing unhealthy, injured, or incapable of producing the character- 
istic rhythmic pumping activity were discarded. Stage of maturity and sex were 
recorded for each of the experimental animals. The worms were distributed by 
size so that each group (experimentals and control) had a representative size 
distribution. Initial weights were taken and experimental animals were placed 
in hypoosmotic test solutions of known salinity. All individuals w r ere then 
weighed at successive 30-min intervals for a period of six hours, or until they were 
removed from the experiment because of rupture (group A). After six hours had 



125 



120- 



t 







| 2345678 

Time after Initial Transfer (Hours) 

FIGURE 4. Time-course of weight changes in Chaetopterus after direct transfers from 901.4 
milliosmoles to: (A) 451.4 milliosmoles (n = 5) ; (B) 681.8 milliosmoles (n == 7); (c) 749.6 milli- 
osmoles (n = 8) ; (D) 802.8 milliosmoles (n == 10); (X) controls, kept at 901.4 milliosmoles 
(n == 11). Star on curve (A) indicates all animals had ruptured, and were removed from experi- 
ment. Arrows at 6 hours indicate transfer back to initial medium. Vertical bars represent one 
S.E. above and below the mean, and are indicated for one curve only, for clarity, as representative 
of the amount of variability encountered in all curves. 



elapsed, the worms were transferred back to seawater of the initial strength and 
weight monitored for an additional 2| hours as before. The data are shown in 
Figure 4 (as absolute per cent body w r eight change) and Figure 5 (corrected for 
controls). Typical variability is indicated by the mean 1 standard error. 

The data in Figures 4 and 5 show that: (1) control worms (remaining under 
acclimated osmotic conditions) slowly lose weight, presumably due to handling; 
(2) worms subjected to hypoosmotic solutions rapidly gain weight, roughly in 
proportion to the degree of osmotic stress ; (3) weight gain is essentially complete 
in 1.5 hours, and is followed by a period of both absolute and relative weight loss; 
and (4) following transfer back to initial salinity, all experimental groups rapidly 



OSMOTIC RESPONSES OF CHAETOPTERUS 



283 



130 







345678 

Time after Initial Transfer (Hours) 

FIOUKE 5. As in Figure 4, corrected for controls. 

lose weight, ultimately dropping below control group values. No significant 
differences were found with regard to sex or state of maturity. 

To examine further the effects of sudden osmotic stress on weight and coe- 
lomic fluid constituents, 30 animals were removed from their tubes, examined, 
weighed and assigned to control (10) and experimental (20) groups as before. 
The experimental animals were transferred to a hypoosmotic solution (ca. 80% 
acclimated seawater) at timed intervals. At 1.5 hours elapsed time all animals 
were weighed, and coelomic fluid samples from five control and five experimental 



f 



o 

s 




0.5 



I 2 3 

Time after Initial Transfer (Days) 



FIGURE 6. Time-course of weight changes (corrected for controls) in Chaetopterus after 
direct transfer from 1100 to 875 milliosmoles. Arrow indicates transfer back to initial medium 
for one group (broken line). For clarity, 1 standard error shown for one group only; n - 
for all points after 6 hours. 



284 



BROWN, BDZIL, AND FRISCH 



120- - 



II5-- 



iio-- 



105" 



I 100 

1 



1 




D 

.c 
5 




I05-- 



100 



105- 



100 




234 
Time after Transfer (Hours) 



FIGURE 7. Experimental results versus behavior predicted from theoretical model. Cross- 
hatch indicates experimental data (mean 1 standard error) ; dotted line from computer solution 
toequations; (1) AC = 0.76,/i = - 0.30; (2) AC = 0.83, = - 0.30; (3) AC = 0.89,0 = - 0.30; 
(4) AC - 0.76, ft = - 0.29; (5 1 AC = 0.83, = - 0.56; (6) AC = 0.89, = - 0.17. 



OSMOTIC RESPONSES OF CHAETOPTERUS 



285 



I20-- 



II5-- 



iio-- 



I05-- 




o 

.c 



O 




I05-- 



100 



2345 
T/'/ne offer Transfer (Hours) 

FIGURE 7 (Continued) 



286 



BROWN, BDZIL, AND FRISCH 



animals (as well as medium samples) were taken. At six hours all remaining 
animals were weighed, and ten experimental animals transferred back to initial 
salinity conditions. One and one-half hours after transfer, five animals were 
removed from the latter group, and coelomic fluid and medium samples taken. 
The weight of the remaining 15 animals (five per group) was monitored at regular 
intervals over a period of four days. The worms were provided with food and 
substitute "houses" (consisting of glass tubes of appropriate diameter) following 
the 12th-hour weighing. Clean water of the proper salinity was added daily. 
Data on the weight changes of the experimental groups (corrected for controls) 
are shown in Figure 6. The results indicate that animals subjected to sublethal 
hypoosmotic stress return to approximately initial weight values after about 24 
hours (Fig. 6, solid curve). In view of the low number of animals involved and 
the variability exhibited, no significance is ascribed to the apparent oscillatory 
behavior of the mean weight around the 100% value. Those animals transferred 
back to the initial salinity lose weight rapidly, ultimately dropping to ca. 90% of 
their starting value (Fig. 6, broken curve). There follows a slow climb in weight 
toward the starting values. 

Coelomic fluid: medium ratios for osmolarity, sodium and chloride, taken 90 
minutes after transfer to new media, are given in Table I. The data show the 
coelomic fluid to be not significantly different from the medium with respect to 
osmotic and ionic concentration. 

DISCUSSION 

Data from the present investigation suggest that Chaetopterus variopedatus 
is an ionic- (Na + ; Cl~) and osmoconformer at sea water concentrations from 1100 
milliosmoles down to a lethal lower limit of approximately 630 milliosmoles. 
This corresponds to a range of 116%-66% seawater (with "100%" seawater 
= 950 milliosmoles). These data are consistent with measurements for other 
marine polychaetes, which indicate that the majority of these annelids are osmo- 
conformers at the higher range of salinities (Oglesby, 1969a). The fact that total 
osmotic pressure closely parallels the sodium and chloride concentrations indi- 
cates that there is only minor contribution (if any) from soluble organic molecules 
to the total osmotic concentration of the body fluids, even at lowered salinities. 
Little is known directly about the organic constituents of Chaetopterus body 
fluids, except that there is no dissolved respiratory pigment (Dales, 1969) and 
that uric acid occurs at a concentration of 43.4 /xAIoles/1 (Wilber, 1948). It is 
concluded that the major osmotic characteristics observed result almost exclu- 
sively from water and inorganic salt fluxes. 



TABLE I. 

Coelomic fluid: medium ratios 1.5 hours after transfer to new medium, 
mean 1 standard error; n = 5 for all values 



Following transfer: 


mOsm (CF:M) 


Na + (CF:M) 


Cl- (CF:M) 


From 1100 to 875 mOsm 
From 875 to 1100 mOsm 


1.02 0.029 
0.99 0.016 


0.99 0.066 
1.01 0.019 


1.06 0.067 
0.99 0.011 



OSMOTIC RESPONSES OF CHAETOPTERUS 287 

The rapid increase in weight (volume) of worms directly transferred to hypo- 
osmotic media presumably reflects an osmotically driven influx of water through 
a readily permeable integument. The volume increase reaches a maximum in 
1.5 hours (Figs. 4, 5, and 6) at which time the coelomic fluid has again reached 
osmotic equilibrium with the medium (Table I). Comparison of initial slopes of 
weight gain (after initial transfer) with weight loss (after back-transfer) also 
clearly indicates that, under the same osmotic gradient, fluid enters more rapidly 
than it leaves. This apparent greater permeability inward than outward has 
been reported for other species by a number of investigators (Adolph, 1936; 
Gross, 1954; JoYgensen and Dales, 1957 ; Oglesby, 1965) although the reason for it 
is not known. 

That some salt efflux has also occurred is indicated from the results of experi- 
ments where hypoosmotically stressed worms were returned to initial salinities 
(Figs. 4, 5, and 6). In every case, worms rapidly lost weight and eventually 
dropped well below the mean weight of the control groups. Weight loss is essen- 
tially complete after l|-2 hours (Figs. 4, 5, and 6), after which there appears to 
be a slow climb toward initial values (Fig. 6). Shrinkage to less-than-original 
volume after transfer back to normal seawater shows that the equilibrium at- 
tained in dilute seawater was reached partly by the loss of solutes (Potts and 
Parry, 1963), 

A return to initial weight (volume) values after transfer to media of various 
salinities is conventionally used as the criterion for judging whether or not a 
marine animal has the ability to control its body volume (Oglesby, 1969a). 
Applying this criterion to data on the time-course of weight changes in hypo- 
osmotically stressed Chaetopterus (Figs. 4, 5, and 6), one must conclude that 
Chatetopterus possesses volume-regulating ability over most, if not all, of its 
viable salinity range. Garrey (1905) is apparently the only previous investigator 
to publish data on the response of Chaetopterus to hypoosmotic media. He 
transferred worms directly from full-strength Woods Hole seawater (A = - 1.82 
C) to: (1) 50% seawater (A = 1.02 C), and (2) "fresh water" (A = - 0.02 C) 
(Garrey, 1905, Table IV). After periods of six hours in 50% S.W. and three 
hours in F.W., the worms increased in weight (by an unstated amount) and ap- 
peared "swollen." In light of results from the present study, it is evident that 
Carrey's stress solutions were in the lethal range for the animals. Forced to base 
his judgment on Carrey's limited data, Oglesby could only conclude that Chaetop- 
terus showed "no" ability to regulate its volume during osmotic stress (Oglesby, 
1969a, Table X, page 251). The volume-regulating ability of Chaetopterus is, 
in fact, quite comparable in magnitude and rate to some of the better-known 
volume-regulating nereids, such as Nereis diversicolor (Beadle, 1937) and N. 
limnicola (Oglesby, 1968). In contrast to these euryhaline species, however, 
Chaetopterus appears to have no (or minor) salt-regulating capabilities and is 
unable to tolerate salinities as low as 50% S.W. 

The mechanism by which an isoosmotic polychaete, such as Chaetopterus, 
"regulates" its volume after transfer to lower salinities is not known. There are 
currently two hypotheses to explain the corrective elimination of fluid in such 
animals. The first suggests that there is an increased efflux of fluid via the ne- 
phridia. The motive force driving this efflux may be produced intrinsically 
(i.e., as a result of increased activity of cilia lining the nephridial tubules) or 



2<SS 



BROWN, BDZIL, AND FRISCH 




MICROMETER 

SYRINGE 



'DIALYSIS "ELASTIC 

MEMBRANE MEMBRANE 



(A) 



6O- 



O /O-i 



5 5^ 






Q. 


^ 


1 1 f 






1 1 1 r " ' 1' ' 1 ' 1 1 1 ' 





456 
TIME (HOURS) 



10 



(B) 

FIGURE 8. (A) "Mechanical engine" with properties of model. See text for details. (B) 
Time-course of volume and pressure changes in chamber B after osmotic gradient established 
at time 0. 

extrinsically (i.e., as a result of increased coelomic hydrostatic pressure caused by 
contraction of the body-wall musculature) (Beadle, 1937, 1943; Smith, 1970). 
A second hypothesis suggests that there is active transport of an ion species 
outward through the general integument. There presumably follows the passive 
diffusion outward of a counter-ion (maintaining electrical neutrality) and the 
passive diffusion outward of water (maintaining osmotic equilibrium). The total 
effect is the net transfer of isoosmotic saline from inside to outside (Prosser and 



OSMOTIC RESPONSES OF CHAETOPTERUS 289 

Brown, 1961). Although these proposecTmechanisms differ markedly, they are 
not mutually exclusive. They are similar in presupposing that an expenditure 
of energy is required to produce the driving force necessary for fluid elimination. 
At present there is virtually no data to support or refute either of these hypotheses 
in detail, and even the studies investigating metabolic rates during volume regu- 
lation have failed to confirm or deny the existence of an energy requirement 
(Oglesby, 1969a). 

One of the conceptual difficulties inherent in these theories relates to the 
negative- feedback character of the phenomenon, since data from many sources 
indicate that in good volume-regulators the amount of fluid efflux is such that the 
worms are returned rather precisely to their initial volumes (given sufficient time 
and sublethal stress). With reference to the active transport hypothesis, for 
example, there are many common systems where changes in salt concentration are 
known to elicit activity of membrane transport systems which act to correct the 
perturbation. When the salt concentration again approaches initial values, 
activity of the transport system is lowered to an appropriate level or stopped. 
Such monitoring of salt levels may be operative in polychaetes capable of both 
ion- and osmoregulation at low external salinities. With regard to isoosmotic 
volume-regulators, however, it would seem that there is a crucial component 
missing namely, the internal salt concentration does not return to prestressed 
values during the following volume decrease. Therefore, a salt concentration 
"signal" to turn off the corrective effector activity (producing fluid efflux) does 
not appear to be present. In contrast, the hypothesis that a type of muscular 
stretch response produces hydrostatic back-pressure at least has the merit of 
suggesting how a mechanically linked "volumestat" servo-mechanism control 
might be achieved. 

Nevertheless, it appears to us that the principle of parsimony has been all but 
ignored by the general assumption that the major route of fluid elimination is via 
the nephridial tubules. The apparent agreement on this point seems particularly 
puzzling since it is also generally acknowleged that the initial water entry occurs 
throughout the entire permeable integumental surface. No one has suggested, 
for example, that the initial rapid increase in volume results primarily from flow 
of water inward through the nephridial tubules. In addition, it is clear that active 
contraction of the body-wall musculature is not the only potential source of in- 
creased internal hydrostatic pressure. Apparently overlooked is the fact that 
the integumental membrane itself is an elastic or visco-elastic element and there- 
fore must, at the very least, supplement muscular contraction in producing in- 
creased hydrostatic pressure. 

We have therefore attempted to get some estimate of the magnitude of fluid 
efflux through the surface membrane of a hypothetical model "worm" in which the 
sole driving force is generated by stretched elastic or visco-elastic elements in 
the surface membrane. To do this we draw upon some of the concepts of non- 
equilibrium thermodynamics and make the following assumptions: (1) for ease in 
calculation, we model our hypothetical "worm" as a cylinder of uniform cross 
section of radius r and an unstressed volume V , for which we can neglect end- 
effects; (2) we assume that the bounding ("integumental") membrane of the 
model worm has no gross discontinuities (representing, for example, open metane- 
phridial tubules) ; (3) we assume that the membrane possesses elastic properties 



290 BROWN, BDZIL, AND FRISCH 

which can be adequately represented by Hooke's Law : 

AP = - G(r - r<>) 

where AP = the change in pressure across the membrane, G -- a constant, and 
the subscript zero denotes the value in the unstressed state; (4) we assume the 
membrane to be more permeable to water than to solutes; (5) we assume the 
membrane is not completely impermeable to solutes; (6) we assume that the 
permeability inward is equal to, or greater than, the permeability outward ; (7) 
we assume that order-of-magnitude estimates for the values for permeability, 
change in radius, change in internal salt concentration, response time, etc. are 
identical with those derived from the experiments with Chaetopterus reported 
above; and (8) we assume no contribution to the system from contracting ele- 
ments or active transport processes. 

Using the basic equations of Kedem and Katchalsky (1958), the volume and 
salt fluxes across the membrane are respectively : 

j == L P (AP -- (rRTAC) (1) 

n == coRTAC + (1 - a)cj (2) 

where L p is the filtration coefficient, a is the reflection coefficient, cu is the solute 
mobility, R is the gas constant, T is the temperature, and AP, AC = C -- C', 
and C MC + O) are, respectively, the differences in hydrostatic pressure, salt 
concentration, and the mean salt concentration across the membrane. Since 
L p and w may take on different values for inflow and outflow across the integument, 
we write 




1 out _ T in 

where ft (= - ; ^- -), is a constant, U(t) is the unit step function, and t* is 
Lp 

the time at which the flow is reversed. Substituting these into (1) and (2), and 
making the change of variables (Crank, 1956) 




The temporal changes of the radius of the cylinder and the number of moles of 
salt therein, N 1 are given by 

dR' 

-- -GR' - [C (1 + N')(1 + R')-'-] 



dR' 

- <r)[C + (1 + 



OSMOTIC RESPONSES OF CHAETOPTERUS 291 

where we have introduced the following scaled variables 



r 



R' = _ i N' - - 1 C = - 

1X I L I, IN - I , , . 1 I, V_ r ., 




laRTCo 1 ^ <ir 

1 - , G = 



w = 



r () aRTCo 1 ' 

2 to(Q) 1 
L p (0) crCo' 



Finding solutions to the above set of nonlinear equations would be a difficult 
task. However, observations made on Chaetopterus indicate that R' and N' do 
not exceed order 10" 1 . Therefore, we may linearize the above, which gives 

+ 2)R' + N' + (1 - C) (3) 



- N'] - (1 - C)& + (1 - a)(l + C) (4) 

Using Laplace transformations, it is a simple matter to find the two character- 
istic frequencies for (3) and (4) and the solution to our problem. We obtain 



R'/(l - C) == e^lsinhxj A, (5) 

where 

A! = = l{[o> - (1 + C)(l - - a) + G + 2J - 4coG}i > 

X 2 = - |[co - (1 + C)(l - cr) + G + 2] < 0. 

Clearly if oiG > 0, R' has the appropriate behavior, since I is a monotone increas- 
ing function of time. If we require that both the initial slope and maximum of 
equation (5) correspond to values obtained from the stress experiments on 
Chaetopterus (Fig. 5), we find 

rrRTC" * 

L p (0)- ^8 X 10- 3 /min. 




with A lt A 2 in the range .156 < A x < .825, -1.04 > A 2 > 1.25. Figure 7 
shows a comparison of the experimentally observed volume changes to those 
predicted on the basis of the calculations from our model. The curves on graphs 
1, 2, and 3 were generated on the assumption that there exists a single L p and /8 
for the integument, and that our best estimate of these is derived from the mean 
value from all three experiments. The predicted curves on graphs 4, 5, and 6 were 
generated on the assumption that the different salinity stresses (and hence the 
degree of stretching of the membrane) influenced the L p and /?, and that our best 
estimate of these values is derived from the mean of each individual experiment. 



292 BROWN, BDZIL, AND FRISCH 

In either case the graphs show remarkably good agreement between the calculated 
and the observed curves, consideration being given to the approximations in the 
former and the variability in the latter. We have also examined the case for a 
symmetrical system (0 == 0), and find that it shows a similar response curve. 
As /3 is decreased from zero, the decay time from the state of maximal strain (to 
zero strain) is increased. 

Using our calculations we can make an estimate of the values of the material 
constants a, G, and o> (a < 0.05, G ~ 1, and & ~ 0.5). Transforming back to 
unsealed variables, we have L p (0) ~ 0.020 cm(atm-min)~ 1 G ~ 0.52 atm, 
co(0) ~ 8.15 X 10~ 8 moles (atm-min-cm 2 )" 1 . Since R'max is of order 5 X 10~ 2 , 
we have *AP,nax of order 1 X 10~ 2 atm, which corresponds to a required maximum 
internal hydrostatic pressure of 7.6 mm of Hg, clearly within the range of measured 
coelomic pressures of polychaetes (Chapman and Newell, 1947 ; Prosser and 
Brown, 1961 ; Wells, 1945 and 1961). The small <r and co(0) obtained are charac- 
teristic of a somewhat unselective membrane through which there is little solute 
flow at constant volume, which is consistent with the characteristics postulated 
for the integument of Chaetopterns. 

The observed volume- regulation can be explained by the following mechanism. 
Initially, solvent flows into the worm as a result of the osmotic pressure difference 
across the membrane. (At the same time there is likely to be a small loss of salt 
to the exterior so that correctly the net volume increase equals volume HO 
inward minus volume salt outward. The initial water influx appears to be so 
rapid, however, that isoosmotic conditions obtain before any great volume of 
salts is lost and therefore it can be largely neglected.) The kinetic energy of 
water entry is transferred to the integument as a result of stretching the elastic 
elements. When the initial osmotic driving force is equalized (i,e.. isoosmotic 
conditions are attained) the energy stored in the distended membrane, via 
increased hydrostatic pressure, forces both salts and water back out through the 
membrane into the surrounding bath. The asymmetric nature of volume- regu- 
lation curves can be explained by the assumptions that although the fluid 
initially entering the animal (osmotically) is virtually pure water, the fluid 
leaving the animal (hydrostatically) must be isoosmotic with the body fluids, 
since preferential loss of water would set up an osmotic gradient in the opposite 
direction again. The rate of fluid (volume) loss will be determined by: (1) the 
hydrostatic pressure, and (2) the permeability of theintegument to both water and 
salt. Since the integument is less permeable to salts, the rate of efflux of iso- 
osmotic saline will be decreased by this slower-moving component. The homeo- 
static nature of the mechanism is obvious. When the worm has returned to its 
original volume, the integument is no longer stretched -the energy stored in the 
elastic element having been completely dissipated in doing the work of expelling 
the fluid. 

Such a mechanism requires no direct input of metabolic energy to the correc- 
tive process. It appeared possible to us, therefore, to construct a "mechanical 
engine" with (at least most of) the assumed properties of the system. Such a 
device, made up of 3 contiguous Incite chambers, is shown in Figure 8A. For 
practical purposes it was necessary to separate the elastic portion of the "integ- 
ument" from the permeable, and thus the configuration of the mechanical 
engine was different than that of our theoretical model system (assumption # 1, 



OSMOTIC RESPONSES OF CHAETOPTERUS 293 

above). However, the properties of the mechanical engine conformed closely 
to those outlined for our theoretical model in assumptions 2, 3, 4, 5, and 8, with 
the dialysis membrane being symmetrical (/3 == 0) an alternative possib'lity in 
assumption 6. Assumption 7 was clearly impossible to achieve and thus the data 
are expressed in absolute units, without direct comparison to those derived from 
Chaetoptems. In practice a solute (sucrose) concentration difference of 0.1-0.5 M 
was initially established across the dialysis membrane, with chamber B (the 
"worm") hyperosmotic to chamber A (the "external medium"). The changes 
in pressure and volume in chamber B could be simultaneously monitored, with 
chamber C serving as a liquid-filled compensation chamber leading to the volume 
capillary. An example of the type of data obtained from the apparatus is shown 
in Figure 8B. The general shapes of the curves conform to the predicted behavior 
and show that such a completely mechanical model system is feasible. 

Several comments are in order concerning the proposed mechanical "volume- 
stat" model. First, such a system will only completely return to its initial volume 
if the membrane acts as a pure Hooke elastic element. We have performed the 
analysis assuming the integument to have both viscous and elastic properties, and 
thus to act as a Maxwell element 

AP = - G (r - r () ) - - X / e~ x(t - T >(r - r u )dr 
L ./o J 

Understandably, the resulting curves vary, depending on the proportion of elastic 
to viscous properties. As G approaches zero (pure viscosity) there is swelling 
but no return. As X approaches zero (pure elasticity) there is swelling followed 
by complete return. When both G and X are greater than zero, there is swelling 
and partial return, depending on the specific values of the constants. 

Secondly, we are fully cognizant that the integument of Chaetoptems is perhaps 
uniquely thin and distensible among those of the larger polychaetes. Previous 
histological studies, for example, have shown that Chaetoptems has, over large 
areas of its body, as few as two cell layers separating the coelomic fluid from the 
outer medium (Bonhomme, 1943; Krekel, 1920; Meissner, 1935; Nicol, 1952; 
Trojan, 1913). In addition, a recent electron-microscope survey study showed 
that Chaetoptems is exceptional in lacking a highly ordered fibrous "cuticle" 
associated with its integumental epithelium (Storch and Welsch, 1970). The 
thinness of the membrane, together with the apparent absence of cuticular fibers 
(a viscous element), is probably crucial to the remarkable agreement between our 
almost assuredly too simplistic theory and the actual experimental results. 

Thirdly, we do not propose thatour mechanical "volumestat" model isthe whole 
answer in isoosmotic volume-regulating worms indeed, it cannot be in those 
animals (possibly a majority) where viscous integumental elements predominate. 
In addition, it would seem unlikely that the evolutionary process would yield only 
a single mechanism for volume-regulation in polychaetes, or possibly for even a 
single species. 

Nevertheless, it is clear that in any worm having a permeable outer integument 
which is even partially elastic, a saline efflux as outlined above will result. It may 
prove profitable to take this particular aspect into consideration in future investi- 
gations into the problem of volume-regulation. 



294 BROWN, BDZIL, AND FRISCH 

We are indebted to Dr. Rimmon C. Fay for his collecting efforts on our behalf, 
and to Mr. Edward Donnelly for his able assistance in maintaining the animals. 
This investigation was supported in part by New York State Research Foundation 
Grant-in-Aid 20-7117 to S. C. Brown; National Science Foundation Grant 
GA-27700 to J. B. Bdzil; and National Science Foundation Grant GP-19881 and 
American Chemical Society Petroleum Research Grant 3519C56 to H. L. Frisch. 

SUMMARY 

1. In Chaetopterits gradually adapted to lowered salinities, coelomic fluid 
osmolarity and sodium and chloride concentrations conform to ambient seawater 
at salinities from 1100 to 630 mOsm. 

2. Experimental animals were unable to tolerate salinities below 630 mOsm 
for extended periods of time. 

3. Worms transferred directly to hypoosmotic stress solutions down to 681.8 
mOsm gained weight (volume) rapidly, reaching a maximum in approximately 
1.5 hours. At this point, the coelomic fluid was isoosmotic with the external 
medium. After 1.5 hours, there followed a decrease in volume, which as shown in 
one experiment, culminated in a return to initial volume values. 

4. Transfers back to full-strength seawater indicate that salt efflux as well as 
water influx occurred. 

5. It is concluded that Chaetopterus is a volume-regulating osmoconformer 
over its viable range of salinities. 

6. Current theories of the mechanism of isoosmotic volume-regulation are 
discussed and a mechanically linked "volumestat" model is proposed. 

7. The behavior of the model system is mathematically analyzed utilizing the 
concepts of non-equilibrium thermodynamics. 

LITERATURE CITED 

ADOLPH, E. F., 1936. Differential permeability to water and osmotic exchanges in the marine 

worm, Phascolosoma. J. Cell. Com p. PhysioL, 9: 117-135. 
BEADLE, L. C., 1937. Adaptation to changes of salinity in the polychaetes. I. Control of body 

volume and of body fluid concentration in Nereis diversicolor. J. Exp. Biol., 14: 56-70. 
BEADLE, L. C., 1943. Osmotic regulation and the faunas of inland waters. Biol. Rev., 18: 

172-183. 
BONHOMME, CH., 1943. L'appareil lumineux de Chaetopterus variopedatus. Recherches histo- 

logiques. Bull. Inst. Oceanogr., Monaco, 843: 1-7. 
CHAPMAN, G., AND G. E. NEWELL, 1947. The role of the body-fiuid in relation to movement in 

soft-bodied invertebrates. I. The burrowing of Arenicola. Proc. Roy. Soc. London 

Series B, 134:431-455. 

CRANK, J., 1956. The Mathematics of Diffusion. Oxford University Press, London, 347 pp. 
DALES, R. P., 1969. Respiration and energy metabolism in annelids. Pages 93-109 in M. 

Florkin and B. T. Scheer, Eds., Chemical Zoology, Volume IV. Academic Press, New 

York. 
GARREY, W. E., 1905. The osmotic pressure of sea water and of the blood of marine animals. 

Biol. Bull, 8: 257-270. 
GOSNER, K. L., 1971. Guide to Identification of Marine and Estuarine Invertebrates. Wiley- 

Interscience, New York, 693 pp. 
GROSS, W. J., 1954. Osmotic responses in the sipunculid Dendroslomum zostericolum. J. Exp. 

Biol., 31:402-423. 
( .' XTER, G., 1961. Some relations of estuarine organisms to salinity. Limnol. Oceanogr., 6: 

182-190. 



OSMOTIC RESPONSES OF CHAETOPTERUS 

J0RGENSEN, C. B., AND R. P. DALES, 1957. The regulation of volume and osmotic regulation in 

some nereid polychaetes. Physiol. Comp. Oecol., 4: 357-374. 

KEDEM, O., AND A. KATCHALSKY, 1958. Thermodynamic analysis of the permeability of bio- 
logical membranes to non-electrolytes. Biochim. Biophys. Ada, 27: 229-246. 
KREKEL, A., 1920. Die leuchtorgane von Chaetopterus variopedatns. Z. Wiss. Zool., 118: 480-509. 
MEISSNER, W. W., 1935. Chaetopterus variopedatus im lichte der Langschen trophocoltheorie. 

Pages 1-156 in H. Fenerborn, W. Meissner and O. Steinbock, Eds., Zoologische Forschun- 

gen Vol. I. Universitatsverlag von Robert Noske, Leipsig. 
NICOL, J. A. C., 1952. Studies on Chaetopterus varicpedatus. I. The light-producing glands. 

J. Mar. Biol. Ass. U. K.,30: 417-431. 
OGLESBY, L. C., 1965. Water and chloride fluxes in estuarine nereid polychaetes. Comp. Bio- 

chem. Physiol., 16: 437-455. 
OGLESBY., 1968. Responses of an estuarine population of the polychaete Nereis limnicola to 

osmotic stress. Biol. Bull., 134: 118-138. 
OGLESBY, L. C., 1969a. Inorganic components and metabolism; Ionic and osmotic regulation: 

Annelida, Sipuncula, and Echiura. Pages 211-310 in M. Florkin and B. T. Scheer, 

Eds., Chemical Zoology, Vol. IV. Academic Press, New York. 
OGLESBY, L. C., 1969b. Salinity-stress and desiccation in intertidal worms. Amer. Zool., 9: 

319-331. 
POTTS, W. T. W., AND G. PARRY, 1963. Osmotic and Ionic Regulation in Animals. Pergamon, 

New York, 423 pp. 
PROSSER, C. L., AND F. A. BROWN, 1961. Comparative Animal Physiology. [2nd Ed.] Saunders, 

Philadelphia, 688 pp. 
SMITH, R. I., 1970. Chloride regulation at low salinities by Nereis diversicolor (Annelida, Poly- 

chaeta). /. Exp. Biol. 53: 75-92. 
STORCH, V., AND U. WELSCH, 1970. Uber die feinstruktur der polychaeten-epidermis (Annelida). 

Z. Morphol. Tiere, 66: 310-322. 
TROJAN, E., 1913. Uber hautdrusen des Chaetopterus vario pedatus . S. B. Akad. Wiss., Wien, 

122: 565-596. 

WELLS, G. P., 1945. The mode of life of A renicola marina L. J. Mar. Biol. Ass. U. K., 28 : 447-464. 
WELLS, G. P., 1961. How lugworms move. Pages 209-233 in J. A. Ramsay and V. B. Wiggles- 
worth, Eds., The Cell and the Organism. Cambridge University Press, Cambridge. 
WILBUR, C. G., 1948. Uric acid in marine invertebrates. J. Cell. Comp. Physiol., 31: 107-110. 



Reference : Biol. Bull, 143: 296-303. (October, 1972) 



OXYGEN CONSUMPTION IN ANTERIOR VERSUS POSTERIOR 
EMBRYONIC SHIELD OF FUNDULUS HETEROCLITUS 

ANNA RUTH BRUMMETT 1 AND WINONA B. VERNBERG 2 

Duke University Marine Laboratory, Beaufort, North Carolina, and the 
Belle W. BarucJi Coastal Research Institute, Columbia, South Carolina 

It has been demonstrated (Brummett, 1968, 1969) that the embryonic shield 
of Fnndnlns has the fate of its various organs and tissues determined early in 
gastrulation. When the anterior shield is excised and implanted into the peri- 
cardial chamber of an older embryo it differentiates brain and eyes; posterior shield, 
similarly handled, forms spinal cord, somitic muscle, gut, and other structures 
appropriate to trunk and tail. Histological studies of these tissues at the time of 
excision reveal no recognizable differences between the cells of the two shield 
regions. How, then, might one elucidate and perhaps characterize the differences 
which the transplantation experiments indicate to be present? 

Promising approaches to the above question might include a comparison of the 
two regions in terms of their fine structure, their biochemistry, and their physiology. 
The experiments reported here represent an attempt to provide information on one 
aspect of the third of these approaches. 

Differences in respiratory activity of various parts of an egg or developing 
embryo have long been accepted as indications of differences in extent and direction 
of differentiation. Barth and Sze (1951) and Sze (1953), for example, measured 
respiration in various parts of the anuran (Rana pipiens} gastrula and established 
respiratory gradients in that embryo which they related to both cellular interactions 
(induction) and differentiation. 

The objective of the study presented here was to determine whether measurable 
differences in respiration exist between the anterior and the posterior embryonic 
shield of the gastrula of the teleost, Fiindulus hetcroclitus. 

MATERIALS AND METHODS 

Eggs of Fiindulus heteroclitus were fertilized in the laboratory. To reduce 
the possibility of genetic variability in developmental rates, gametes were obtained 
from one male and one female for each set of experiments. Following fertilization, 
the eggs were kept at room temperature (approximately 24 C). When the eggs 
reached the desired stage of gastrulation [Oppenheimer (1937) stages 12, 14 and 
16 which correspond to Armstrong and Child (1965) stages 15, 18, and 20], 
they were washed in 3% formalin-sea water to reduce the possibility of contamina- 
tion, rinsed in filtered sea water, and dechorionated in sterile Tyrode solution (with- 
out bicarbonate) according to methods previously described (Brummett, 1968). 

1 Present address: Department of Biology, Oberlin College, Oberlin, Ohio 44074. 

2 Present address: Belle W. Baruch Coastal Research Institute, University of South 
Carolina, Columbia, South Carolina 29206. 

296 



TISSUE RESPIRATION IN TELEOST GASTRULAE 



297 



Operating in sterile Tyrode solution, portions of the embryonic shield were care- 
fully excised with finely sharpened watchmaker's forceps (Brummett, 1968, 1969). 
Taking care to avoid transferring yolk or periblast along with the tissues, the ex- 
cised tissue was picked up with a micropipette and placed in a small amount of 
Tyrode solution in a spotting plate depression. As quickly as possible, each piece 
was then transferred from the spotting plate to Cartesian diver respirometers in 
a medium of sterile Tyrode solution. The total volume of the divers ranged from 
10-13 p.1. All metabolic rate determinations were made at 25 C for a period 
of approximately two to two and one-half hours. 

Operations for weight determinations were performed as above in Tyrode solu- 
tion. The excised shield parts were rinsed briefly in distilled water to remove most 
of the balanced salt solution, transferred to a preweighed piece of plastic wrap 
(Cutrite brand) or aluminum foil, dried overnight at 110 C, and weighed on a 
microtorsion balance having a sensitivity of 2 /ig. 



Oxyaen consumption expressed as y ul O z per hour 



dry weight x 10 



Posterior Embryonic Shield 
Mean. 'Ran^g No.D^t 



Anterior Embryonic Shield 
No. Det. Ran^ Mean 



CO 

<d 

Q) 

<J 

C 



* 

tf 
d 
o 



2.98 1 



4.2210. 




1.99 0.04 



1.84- 



P12>A12 



OT 
(J 



s- 
o 



d 
u 



P14>A14 



Plfc>>A16 



P16 > P12 > P14 > A12 > AI4 > A16 



FIGURE 1. Summary of Oz consumption data for anterior i>crsns posterior embryonic 
shield in three stages of development: early gastrula (stage 12), late gastrula (stage 14), and 
tail bud stage (16). The number of individual determinations made, the range of measurements 
obtained, the means standard errors for each pool, and significant increases and decreases 
are indicated. 



298 



A. R. BRUMMETT AND W. B. VERNBERG 



I- 
9 
8 
7 

"9 6 

M 

Q^j 5 

1 



c 
o 

'i 2 

g 

3 
(O 

o 
u 



o 



POSTERIOR SHIELD 



-*- 



ANTERIOR SHIELD 



STAGE 12 
EARLY bASTRULA 



STAGE 14 
LATE GASTRULA 



STAGE 16 
TAIL BUD 



FIGURE 2. Graph comparing O 2 consumption data for anterior (H) versus posterior (O) 
embryonic shield of early gastrula (stage 12), late gastrula (stage 14), and tail bud stage (16) 
of Fundulus hcteroclitus embryos. Standard errors are indicated in each case. Posterior 
shield measurements are significantly higher than anterior shield measurements at each stage 
of development and the difference increases as the embryo progresses from early gastrula 
to tail bud. 



Results are expressed as /A O 2 /hr per /xg dry weight 10 3 . Significance of 
difference of means was determined by the method for small samples given in 
Simpson, Roe, and Lewontin (1960). 

RESULTS 

Results are summarized in Figures 1 and 2. The metabolic rate of the posterior 
shield was significantly greater than that of the anterior shield in all three stages 
of development studied. Furthermore, these differences tended to increase as 
gastrulation proceeded: at early gastrula (stage 12) the metabolic rate of the 
anterior shield was 28% less than that of the posterior shield, at mid-gastrula 
(stage 14), 33% less, and immediately following blastopore closure (stage 16), 
64% less. The level of oxygen uptake in the anterior shield tended to decrease with 
age ; the rate decreasd 18% (significant at <1% level) between stages 12 and 14 and 
approximately 8% (not significant) between stages 14 and 16. In contrast, oxygen 
consumption in the posterior shield decreased 11% (not significant) between 
stages 12 and 14 but increased approximately 42% between stages 14 and 16 
(significant at the <1% level). 



TISSUE RESPIRATION IN TELEOST GASTRULAE 299 

DISCUSSION 

Since injured cells will cytolyze, and cytolysis itself will cause changes in O 2 
consumption, it is important to point out that explants of the early Fundulus embryo 
can be maintained in Tyrode solution for two days or longer without apparent 
damage to the cells (Brummett, Haynes, and Pillsbury, unpublished data). The 
duration of these O 2 consumption experiments was usually less, and never more, 
than three and one-half hours from the time of excision of the embryonic regions 
under consideration to termination of the experiment. It seems reasonable to 
assume then, that the health of the explanted tissues is not a problem in these 
experiments. 

Anterior embryonic shield 

The progressive decrease in oxygen consumption in anterior shield fragments 
as the shield progresses from stage 12 to stage 16 is not consonant with the notion 
that metabolic rate and hence energy requirement increases as differentiation pro- 
gresses along a time axis. One is forced to consider other changes in the embryo 
which might explain the data. One possibility which suggests itself to the authors 
involves the idea of a progressive decrease in the movement of cells in the anterior 
shield accompanied by a decrease in metabolic rates during this developmental 
period. Earlier grafting experiments (Brummett, 1969) indicate that at stage 12 
the anterior embryonic shield is composed of presumptive forebrain and retinal 
tissue. Such tissue, excised and implanted into the pericardial chamber of an 
older embryo, had differentiated brain in 100% and retina in 27% of the sectioned 
grafts. However, 37% of the donors of that tissue developed in an apparently nor- 
mal fashion and only 13% were totally lacking forebrain and eyes. These results 
suggest that cells are in the process of migrating into the anterior shield during and 
perhaps subsequent to early gastrula (stage 12). Such an interpretation is in 
agreement with the data reported by Ballard and Dodes (1968) on the trout embryo 
where disengaged cells below the cellular envelope are described as moving into the 
anterior shield from the central region of the blastodisc during the early stages of 
epiboly: "As seen from below and confirmed in sagittal sections, the anterior and 
lateral edges of the gathering embryonic shield, even at six days, still grades off 
gently and smoothly into the thinned areas of the blastodisc. The convergence 
movements which bring together the prospective forebrain area of the shield become 
conspicuous at seven days, whereupon this part of the shield develops sharp 
boundaries. The outlying thinned area is by then two cells thick, consisting of 
the cellular envelope and one layer of inner cells" (Ballard and Dodes, 1968, 
page 77). 

Extensive study of developing Fundulus embryos suggests that, except for the 
time factor (the smaller egg of Fundulus develops much more rapidly than the 
trout egg), the quoted description of anterior shield formation in the trout is 
consonant with the situation in the embryo of F. hcteroclitus. It seems reasonable 
to suppose, then, that in early stages of gastrulation there would be considerable 
movement of cells comprising the early anterior embryonic shield and that, as the 
shield becomes more definitely formed, the movement would decrease and total 
oxygen consumption might decrease concomitantly. Significant decrease in cell 
movement would presumably obscure any increase in metabolic rate associated with 
progressive differentiation in the stages under consideration here. 



300 A. R. BRUMMETT AND W. B. VERNBERG 

Posterior embryonic shield 

The increase in oxygen consumption in the posterior embryonic shield during 
gastrulation is perhaps more difficult to explain. It is almost certain that cells are 
actively moving into the posterior shield from the lateral shield at both stage 12 and 
stage 14 (Brummett 1954), and perhaps the amount of activity is essentially the 
same at these two stages. It seems unlikely, however, that such movement would 
be greater at stage 16 after blastopore closure and the formation of the definitive 
tail bud. Differentiation has no doubt proceeded further at stage 14 than at stage 
12 and still further at stage 16, but in view of the anterior shield picture it would 
seem presumptuous to attribute the significant increase in oxygen consumption to 
what would appear to be minor differentiative changes. The question of differential 
growth comes to mind, but increased mitotic activity in the tail bud blastema must 
also be set aside as an acceptable explanation of the increase in oxygen consumption 
in this tissue if one accepts as pertinent Pasteels' (1943) results which revealed 
essentially no differences in mitotic activity when the blastoporal region w*as com- 
pared with the anterior shield region of the trout embryo. However, this possibility 
will be considered further below. 

Comparison of anterior versus posterior shield 

The phenomenon of early morphogenesis of brain, common in vertebrate em- 
bryos, might lead one to expect earlier and more extensive differentiation in the 
anterior region of the embryonic shield and hence, perhaps, a higher metabolic rate 
there than in the posterior shield. Such an expectation is not consistent with the 
results of these experiments, however, since the posterior shield exhibits a higher 
rate of oxygen consumption at all three of the stages measured, and the difference 
increases as gastrulation proceeds. The data suggests, then, that the anterior 
shield is not significantly ahead of the posterior shield along the differentiative 
pathway. This interpretation is reasonable in the light of the results of grafting 
experiments (Brummett, 1968, 1969) which reveal similar time scales for graft 
differentiation whether the tissue is derived from the anterior or posterior shield. 
Both donors and grafts of the earlier experiments also demonstrate that even at stage 
12, the posterior shield is determined to form trunk and tail structures and the 
anterior shield is determined to form primarily forebrain, midbrain, and associated 
structures ; presumptive hindbrain is intermediate in position and may be excised 
with either an anterior or a posterior stage 12 graft. In other words, determined 
cells appropriate to the entire axis are already present in the early embryonic shield. 
There is no evidence that presumptive head structures are present first (and 
hence are differentiatively older), and that presumptive trunk and tail structures 
enter the shield later as epibody progresses and the shield elongates by virtue of the 
convergence of cells from the lateral shield and germ ring. With respect to the 
time axis of differentiation, then, anterior and posterior shield are, in all probability, 
differentiating simultaneously ; i.e., no one area exhibits a precocity of differentiation 
when compared with other areas of the developing embryo. 

The preceding discussion is germane to that aspect of the results which shows 
that the rate of oxygen consumption in the anterior shield is not higher than in the 
posterior shield. It does not, however, respond to the opposite side of the question : 



TISSUE RESPIRATION IN TELEOST GASTRULAE 



301 



i.e., why is oxygen consumption in the posterior shield significantly higher than in 
the anterior shield? 

It was suspected that differential growth rate might possibly be the source of 
the observed difference. With this idea in mind the data recorded in the earlier 
grafting experiments were reviewed, and the time at which obvious growth was first 
observed and recorded for individual living grafts was noted. When this informa- 
tion for the two series of experiments was compared, it became apparent that in 
the majority of those cases in which such information was recorded, the posterior 
grafts exhibited an increase in size earlier than did the anterior shield grafts. 
Indeed, during the first 24 hours following transplantation, growth was noted in 
7\% (50 of 71 cases) of the posterior grafts as compared with only 8% (3 of 37 
cases) of the anterior shield grafts (see Table I). Since the earlier experiments 
were not designed with this particular question in mind, the records are not as 
complete as one might wish, but the data agree with the notion that differential 
growth rate may contribute to higher oxygen consumption in the posterior shield. 

TABLE I 

Tabulation of growth data (the time when increase in size of graft was first noted} recorded in earlier 

experiments for anterior and posterior embryonic shield grafts. In general, posterior grafts 

exhibited increase in size earlier in the post-operative period than did anterior 

shield grafts 



Anterior embryonic shield grafts 


Hrs 


Posterior embryonic shield grafts 




Totals 


St. 12 


St. 13 


St. 14 


St. 15 


opera- 


St. 12 


St. 13 


St. 14 


St. 15 


Totals 






# (7 ) 


# (%) 


# (%) 


# (%) 


# (%) 




# (%) 


* (%) 


# (%) 


#(O7 1 
\ /Of 


# (%) 













- 








6-12 


1 (ID 


7 (37) 


9 (28) 


4 (36) 


21 (30) 


i 


8% ) 


3 (8) 





3 (19) 








18-24 


5 (55) 


4 (21) 


14 (44) 


6 (55) 


29 (41) 


j '" 




2 (5) 


I (8) 


1 (6) 








30-36 


2 (22) 


2 (11) 


6 (19) 





10 (14) 






10 (27) 


2 (15) 


4 (25) 


1 (20) 


3 (100) 


42-48 


1 (11) 


5 (26) 


1 (3) 


1 (9) 


8 (11) 






6 (16) 


2 (15) 


3 (19) 


1 (20) 





54-60 





- 


2 (6) 


- 


2 (3) 






16 (43) 


8 (62) 


5 (31) 


3 (60) 





66-72 





1 (5) 





- 


1 (1) 






37 


13 


16 


5 


3 




9 


19 


32 


11 


71 





Mitotic counts should be made to substantiate this interpretation. As was men- 
tioned earlier, however, mitotic counts on the trout embryo by Pasteels (1943) 
produced negative results. 

It is of interest to note that in her studies of metabolic gradients in teleost 
embryos, Hyman (1921) found that in Fundiilus heteroclitus the pattern of dif- 
ferential susceptibility to toxins always began at the posterior end of the embryonic 
axis and proceeded anteriorly. A second susceptibility gradient began subsequently 
at the anterior end of the axis. Hyman does not provide a great deal of data 
but what is presented is in agreement with that presented in this paper. Hyman 
interprets her data to mean that the region of high activity at the posterior end of 
the embryonic axis reflects a growing point which is responsible for the laying 
down of the greater part of the Fnndulus embryo. 

Comparison of these results with similar experiments on the amphibian ga-strula 

Using essentially the same methods described in this paper, Sze (1953) measured 
oxygen consumption in various portions of the early gastrula of the frog, Rana 



302 A. R. BRUMMETT AND W. B. VERNBERG 

pipicns. The results of those experiments indicate that oxygen consumption in the 
frog embryo is less than that in the Fundulus embryonic shield by a factor of ten. 
This is not surprising since the cells of the early amphibian embryo contain a con- 
siderable amount of stored yolk which is, presumably, metabolically inert. The 
cells of the teleost embryo, on the other hand, contain little or no stored yolk but 
appear to absorb it as needed through the periblast membrane which separates the 
developing embryo from direct contact with the yolk. 

In this study Sze (1953) found that the presumptive neural regions of the 
early frog gastrula exhibited a higher rate of oxygen consumption than did the 
presumptive chordamesoderm. Deletion-grafting experiments (Brummett, 1968) 
demonstrate that the posterior shield tissue in Fundulus is not limited to chordameso- 
derm but consists, rather, of cells which are already determined to form all tissues 
appropriate to posterior trunk and tail. The regions of the frog gastrula measured 
by Sze (1953), then, are not directly comparable to the teleost tissues used in the 
experiments reported here. 

In another series of experiments in which parts of the frog gastrula in various 
combinations were subjected to respiration measurements, Barth and Sze (1951) 
attributed increased oxygen consumption to inductive interactions between "or- 
ganizer" and presumptive neural plate or presumptive epidermis. Although the 
teleost posterior embryonic shield has for many years been homologized with the 
dorsal lip region of the early amphibian embryo, convincing evidence that it is an 
"organizer" region is lacking. The posterior embryonic shield of the Fundulus 
embryo, however, does contain a wider variety of presumptive tissues than does 
the anterior shield as revealed by the differentiation of grafts from these two regions 
(Brummett, 1968, 1969). Nothing is known concerning possible inductive inter- 
actions among these various components of the posterior shield in the teleost 
embryo, but of possible significance is the fact that presumptive notochord appears 
to be present in the posterior embryonic shield from stage 12 on (Brummett, 1968). 
In contrast, notochord never differentiated in anterior shield grafts of stages 13, 
14, and 15, and was found to be present only in those stage 12 grafts which had 
included more than half of the very early shield (Brummett, 1969). Perhaps, then, 
the results of the experiments reported here can best be explained in terms of energy- 
consuming cell interactions (including induction?) which may be significantly 
greater in the posterior than in the anterior shield at all stages of gastrulation, which 
may increase in the posterior shield as the embryo goes from stage 12 to stage 16, 
and which may decrease in the anterior shield during the same period. 

It is felt that the experimental data presented in this paper is convincing evidence 
that measurable physiological differences exist in the posterior versus the anterior 
embryonic shield at a stage when morphological differences in the cells, at the level 
of magnification possible with light microscopy, is lacking. Electron microscope 
studies on these tissues are in progress in the hope that they might provide further 
elucidation of the question of differences. Cell movements, cell proliferation, cell 
interactions, and cell differentiation are undoubtedly occurring simultaneously in 
the tissues which were excised and measured in these experiments. These various 
aspects of cell activity no doubt vary quantitatively in different regions of the 
embryo and in the same region at different stages of development. They may also 
vary considerably in their energy requirements. It is difficult, then, to delineate 



TISSUE RESPIRATION IN TELEOST GASTRULAE 303 

accurately the causal factors responsible for the differences in oxygen consumption 
demonstrated by these experiments. We have attempted in the discussion above, 
however, to consider the possible contribution of each of these important aspects of 
cell activity to the differences in O 2 uptake obtained in these experiments, and we 
have tried to interpret the results in the light of what is known about the teleost 
embryo at this stage of development. 

SUMMARY 

1. Experiments were designed to determine whether measurable differences in 
respiration exist between the anterior and posterior embryonic shield of the gastrula 
of Fundulus heteroclitus. 

2. Anterior and posterior embryonic shields were carefully excised from embryos 
of early gastrula, late gastrula, and post-blastopore-closure stages. Using Cartesian 
diver microrespirometers (10 to 13 /A), oxygen consumption of each individual 
explant was measured at 25 C over a period of two to two and one-half hours. 

3. Oxygen consumption data, expressed as jul O 2 per hour per /*g dry weight 
10~ 3 , for the two embryonic shield regions at the three stages of development are 
compared. 

4. Posterior shield was found to exhibit a significantly higher rate of O 2 uptake 
than anterior embryonic shield at all three stages of development. 

5. Oxygen uptake appears to increase in the posterior shield as development 
proceeds from early gastrula to closure of the blastopore ; anterior shield exhibits a 
concomitant decrease. 

6. The results are discussed in light of what is known about cell movements, 
cell proliferation, cell interactions, and cell differentiation in the teleost embryo 
and are compared with similar experiments on amphibian embryos. 

LITERATURE CITED 

ARMSTRONG, P. B., AND J. S. CHILD, 1965. Stages in the normal development of Fundulus 

heteroclitus. Biol Bull, 128: 143-168. 
BALLARD, WILLIAM W., AND LANCE MEREDITH DODES, 1968. The morphogenetic movements 

at the lower surface of the blastodisc in salmonid embryos. /. Exp. Zool., 168 : 67-84. 
BARTH, L. G., AND L. C. SZE, 1951. The organizer and respiration in Rana pipicns. Exp. 

Cell Res., 2 : 608-614. 
BRUMMETT, A. R., 1954. The relationships of the germ ring to the formation of the tail bud in 

Fundulus as demonstrated by the carbon marking technique. /. Exp. Zool., 125 : 

447-486. 
BRUMMETT, A. R., 1968. Deletion-transplantation experiments on embryos of Fundulus 

heteroclitus. I. The posterior embryonic shield. /. Exp. Zool., 169: 315-334. 
BRUMMETT, A. R., 1969. Deletion-transplantation experiments on embryos of Fundulus 

heteroclitus. II. The anterior embryonic shield. /. Exp. Zool., 172: 443-464. 
HYMAN, LIBBIE H., 1921. The Metabolic Gradients of Vertebrate Embryos. I. Teleost Em- 
bryos. Biol. Bull, 40 : 32-73. 

OPPENHEIMER, J. M., 1937. The normal stages of Fundulus heteroclitus. Anat. Rec., 68: 1-15. 
PASTEELS, J., 1943. Proliferations et croissance dans la gastrulation et la formation de la queue 

des Vertebres. Arch. Biol., 54: 2-51. 
SIMPSON, G. G., A. ROE AND R. C. LEWONTIN, 1960. Quantitative Zoology. Harcourt, Brace 

and Company, New York, 440 pp. 
SZE, L. C., 1953. Respiration of the parts of the Rana pipiens gastrula. Physiol Zool, 26 : 

212-231. 



Reference: Biol. Bull.. 143: 304-316. (October, 1972) 



TEMPERATURE, WATER, AND RESPIRATORY REGIMES OF AN 

AMPHIBIOUS SNAIL, POMACEA URCEUS (MULLER), 

FROM THE VENEZUELAN SAVANNAH 

ALBERT J. BURKY, 1 J. PACHECO, AND EUGENIA PEREYRA 

Institute de Zoologia Tropical, Facultad de Ciencias, Universidad Central de Venezuela, 

Caracas, Venezuela 

It has long been known (Troschel, 1845; Pelseneer, 1895; Prashad, 1925, 
1932) that ampullariid or "pilid" snails possess "amphibious" respiratory struc- 
tures, one part of the mantle cavity containing a ctenidium (the characteristic mol- 
luscan gill) and another part being modified as a gas-filled lung cavity. This 
double adaptation for aquatic and aerial respiration in Pomacea urccus is probably 
most important during the rainy season (Burky and Burky, in preparation). 
Respiration is amphibious in both its structural (Andrews, 1965) and its behavioral 
(Burky and Burky, in preparation) aspects. In addition, the annual sequence of 
rainy and dry seasons of the Venezuelan plains dictates an amphibious way of life 
for Pomacea urceus (Burky, in preparation) . 

Terrestrial snails face particular problems of temperature regulation and 
water balance since their moist bodies are exposed during activity (Howes and 
Wells, 1934a, 1934b; Hogben and Kirk. 1944; Dainton, 1954a, 1954b; Russell 
Hunter, 1964; Machin, 1964, 1965, 1966; Cloudsley-Thompson. 1968, 1970; 
Vernberg and Vernberg. 1970). The relationship of body temperature to en- 
vironmental temperature has been reported for intertidal (Lewis, 1963; Fraenkel, 
1968; Grainger, 1968; Davies, 1970; Newell, Pye, and Ahsanullah, 1971; 
Vermeij, 1971 ; and others) and for other terrestrial gastropods (Hogben and Kirk, 
1944; Dainton, 1954a; and others). In deserts and in tropical areas with periodic 
dry seasons there are additional problems of high temperatures and limited water. 
Under arid conditions the body temperatures of the pulmonates HeUcella inrgata 
(Pomeroy, 1968) and Trochoidea seetseni (Yom-Tov, 1971a) are a function 
of their position in the temperature gradient between air and ground as they 
aestivate attached to vegetation above the ground. When the pulmonate Sphmctero- 
chila boissieri is dormant on the ground surface, body temperature is primarily a 
function of slow conduction from the substrate and the transfer of body heat to the 
air (Yom-Tov, 1971a; Schmidt-Nielsen, Taylor, Shkolnik, 1971). For these 
pulmonates, the rate of water loss is too low to be of value for evaporative cooling. 
In desert snails the high reflectivity of light-colored shells is important in reducing 
the absorption of solar radiation (Yom-Tov, 1971a; Schmidt-Nielsen et al, 1971). 
Some snails wait out severe climatic periods in the ground (Pain, 1950; Meenakshi, 
1964; Coles. 1968 ; Pomeroy, 1968 ; Yom-Tov, 1971a, 1971b; Schmidt-Nielsen et al, 
1971 ; and others). In aestivating ampullariids, Pomacea lineata can tolerate a loss 

1 Present address : Department of Biology, Case Western Reserve University, University 
Circle, Cleveland, Ohio 44106. 

304 



TEMPERATURE REGIME IN POMACEA 



305 



of over 50% of its tissue weight (Little, 1968) while Pila virens dies when 30% 
of its tissue water has been lost (Meenakshi, 1964). Also, metabolism during 
aestivation in snails belonging to the family Ampullariidae has received attention. 
The Indian ampullariid, Pila virens, has been shown to be anaerobic (Meenakshi, 
1956, 1957, 1964) and the African ampullariid, Pila ovata, has been shown to be 
aerobic ( Visser, 1965 ; Coles, 1968, 1969) during aestivation. Such differences raise 
questions about metabolism as well as water economy and temperature regulation 
in a neotropical ampullariid like Pomacea urceus. 

Since life history, growth, and biomass production (Burky, in preparation), 
and buoyancy changes as related to respiratory behavior (Burky and Burky, in 
preparation) were being studied for Pomacea urceus, it was decided to investigate 
the temperature and water regimes during aestivation and to measure oxygen con- 
sumption in active and aestivating snails. The adaptive significance of the data 
on temperature, water loss, and oxygen consumption is discussed in relation to the 
annual dry season and to the other existing information on Pomacea urceus (Burky, 
in preparation ; Burky and Burky, in preparation). 



u 

o 



o> 

Q. 

O 



a 
a> 



40- 



30- 



CL 

E 
a> 



a> 

_c 
CO 



20- 




t 



I " 



O 



20 30 40 

Temperature of Exposed Wooden Surface 



5 



FIGURE 1. Shell temperature near the operculum of Pomacea urceus is plotted against the 
temperature of the exposed wooden surface; snails are experimentally exposed to direct sun 
during aestivation. The isothermal line is given for reference. The stylized diagram gives 
the position of an experimental snail in relation to sun and wooden surface. The closed 
triangles represent the positions of the thermistor probes for the plotted data. The open 
triangles give the positions of thermistor probes recording the temperatures of shaded air 
and upper exposed shell surface. For further details, see text. 



306 



BURKY, PACHECO, AND PEREYRA 



50 



o 




30 



35 40 

Oven Temperature 



45 



50 



FIGURE 2. Body temperature of Pomacea urceus (closed circles) with 95% confidence 
intervals and the temperature of water in an open beaker (open squares) are plotted against 
oven temperature. The isothermal line is given for reference. For further details, see text. 

MATERIALS AND METHODS 

Pomacea urceus was collected in the plains (Los Llanos) region of Venezuela. 
Field studies were carried out in the local area known as El Estero de Camaguan 
near the village of Camaguan in Guarico State. Laboratory experiments on tem- 
perature and oxygen consumption were carried out on aestivating and active snails 
collected during the dry and rainy seasons, respectively. 

Snails were experimentally exposed to the tropical sun to record their response 
to the absorption of solar radiation. Aestivating adult snails were placed (aperture 
opening downward) on a gray wooden surface on a roof terrace of the laboratory in 
Caracas. Experiments were set up in the morning before the sun was high enough 
to directly illuminate the experimental area (initially the snails were in the shade). 
In this way the snails were gradually exposed to the direct tropical sun. During the 
day the major influences on solar radiation were the passage of clouds and the 
angle of the rays as the position of the sun changed. Temperatures of shaded 
air, exposed wooden surface about 25 cm from the snail, upper exposed shell sur- 



TEMPERATURE REGIME IN POMACEA 307 

face, and lower shaded shell surface (columella next to the operculum) were re- 
corded at 20 minute intervals with YSI thermistor probes. The position of these 
probes is indicated in Figure 1. The shell temperature next to the operculum 
was chosen as an index of body core temperature because this area was shaded ; it 
was not in contact with the wooden surface; it was adjacent to a large volume of 
tissue; and the probe could be taped to the shell without disturbing the snail or 
obstructing the movement of the operculum. During aestivation the operculum of 
a large snail can be withdrawn 3 to 6 cm from the aperture edge of the columella 
area. At the end of some experiments a small hole was made in the shell (using 
a ballpeen hammer) and core temperature was recorded (less than 30 seconds needed 
for this manipulation) with a hypodermic thermistor probe. These core tem- 
peratures can then be compared to temperatures taken next to the operculum. The 
core temperatures of 12 snails average 1.2 C higher than the columella shell tem- 
perature (core index) of Figure 1. 

Snails were maintained at controlled temperatures so their body temperature 
could be compared with the temperature of water having a free surface. Groups 
of five to eight adults were maintained for four hours or longer at oven temperatures 
of 25, 35, 40, 45, and 50 C. Shell temperature and temperature of water in an 
open beaker (100 ml roughly equivalent to snail tissue volume) were recorded 
at 20 minute intervals until shell temperature was constant for three successive 
readings. Body core temperature was taken at the end of each experiment ; mean 
values with 95% confidence limits are given in Figure 2. 

Field temperatures were taken before the end of the dry season and recorded 
with YSI thermistor probes. The body core temperatures were taken only after 
all other values had been recorded. The snails were removed from their positions 
of aestivation, a hole made, and the thermistor probe inserted (less than one minute 
needed for this manipulation). The soil moisture content was measured with a soil 
moisture meter (O.S.K. 450 Riken Moisture Meter, Model R-l-1 Moisture Indi- 
cator), a calibrated conductivity determination. These field values are summarized 
in Table I. 

Weight loss during aestivation was measured as an index of tolerance to water 
loss. For weighing experiments snails were initially maintained in large outside 
tanks and fed lettuce. Active adults (males and females) were removed from these 
tanks, drained, weighed, and placed in open cardboard boxes to initiate aestivation. 
Snails were maintained in the laboratory at room temperature, generally between 20 
and 25 C, and weighed at varying intervals. The snails were usually weighed 
over a period of five days, so a mean date was determined for each weighing. A 
long term and a short term experiment were started in January 1969 and in 
February 1970, respectively. Each snail's weight is expressed as a percentage of 
its weight on the first day of aestivation. The mean percentage and 95% confidence 
limits have been computed for the snails of each weighing. 

The oxygen consumption of aestivating and active snails was measured at 30 C 
using two plexiglass chambers (1.6 liter) and Warburg manometers (KOH was 
used for CO 2 absorption). One to four aestivating snails could be placed in a 
chamber while only one active snail could be measured in an experiment. Thirty 
minutes to an hour was allowed for equilibration and one chamber was used as a 
thermo-barometer. Readings were taken at about 30 to 60 minute intervals. After 



308 BURKY, PACHECO, AND PEREYRA 

each experiment the shell length, whole animal live weight, body wet weight (no 
shell), and body dry weight (100 C in an oven until constant weight) were 
determined (Table II). 

RESULTS 

The relationship of columella shell temperature near the operculum (index of 
core temperature) to exposed wooden surface temperature (25 cm from shell) 
is shown for 25 individual adult snails (males and females, shell lengths 94 to 
129 mm) in Figure 1. The temperatures near the operculum (core index) for 
exposed wooden surface temperatures above 24-25 C (Fig. 1) were recorded 
after the snails were no longer in the shade. Those points above the isothermal 
line were recorded after clouds had obstructed solar radiation and indicates that 
the wooden surface cools faster than the shell near the operculum (core index). 
Throughout these experiments the temperature of the upper exposed shell surface 
(data not given, see diagram of Figure 1) were higher than the temperatures of the 
exposed wooden surface, due to heat absorption by the dark brown to black coloring 
of the snail shells. Shaded air temperature (data not given, see diagram of Figure 
1) were always 8-10 C lower than those of the shell near the operculum (core 
index) at higher temperatures (above 35 C). This indicates some heat flow from 
the snail to the air. During these experiments on aestivating adults, opercular 
movements (exposure of mantle tissue to air) were commonly observed, particularly 
at higher temperatures when evaporative cooling can be assumed to be important. 
All snails survived these experiments. The data of Figure 1 indicates regulation 
of body temperature, generally below 41 C when animals are experimentally 
exposed to direct solar radiation. 

Figure 2 gives the results of experiments at controlled air temperatures in an 
oven. The higher body temperatures at 25 C in the oven might be a factor of 
metabolic heat production. The body core temperatures at oven temperatures of 
35, 40, and 45 C were about 0.5 C higher than the water temperatures of open 
beakers. Snails at 40 C remained in good condition (opercular movements ob- 
served) while the bodies of those at 45 C were extended to the edge of the aperture 
opening with foot tissue visable. The snails at 45 C didn't retract rapidly when 
touched. The bodies of the snails at 50 C were extended beyond the edge of the 
shell aperture ; they were either moribund or dead after a few hours. This condition 
is reflected in the large variation in the body temperature of snails at 50 C 
(Fig. 2) with a mean core temperature which is essentially the same as the tem- 
perature of water in an open beaker. These experiments at controlled temperatures 
indicate an upper lethal temperature between 40 and 45 C and show that evapora- 
tive water loss is responsible for lowering body temperature (Fig. 2). 

Four groups of 25 aestivating spat (mean shell length about 10 mm and mean 
weight about 0.3 g) were exposed to the mid-day tropical sun in April 1970. 
Within 30 minutes all had fluid bubbling from the edges of their opercula. At the 
end of two hours they were submerged in water and all were dead. Another group 
(150 spat) was not exposed to the sun and all were active within one hour of 
submersion in water, some within three minutes. Significantly, adults were exposed 
to the sun for many days without mortality. Exposure in the field is discussed 
below. 



TEMPERATURE REGIME IN POMACEA 



309 



T 



GROUND 



10cm 




SURFACE 



2cm 

IM 



6 cm 



M 2 



T6 



FIGURE 3. The position of a female Pomacca nrcciis (stylized shell) with eggs during 
aestivation in the field. Ti to T (C) are reference points for temperatures given in Table I 
and correspond to shaded air, ground surface, soil (10 cm depth) about 25 cm from snail, 
exposed shell surface, soil beneath snail, and body core temperature respectively. Mi, M 2 , and 
M 3 are reference points for the soil moisture content values of Table I. The depths, shell, 
and eggs are drawn to scale. 



TABLE I 

Temperature and moisture regimes for Pomacea urceus during aestivation in the savannah. 
perature (C T\ to JT 6 ) and soil moisture content (weight ratio M\ toM-.\\ 
values correspond to the reference points of Figure 3 



Tern- 





Morning (8:15-9:30) 


Mid-day (12:30-14:45) 




May 2. 1970 


May 1, 1970 








95% 






95% 




Range 


Mean 


confidence 


Range 


Mean 


confidence 








limits 






limits 


Tj 


31.5-34.0 


32.71 


1.128 


35.0-40.0 


38.17 


1.231 


T 2 


32.5-38.5 


35.57 


2.511 


42.5-54.5 


49.61 


3.012 


T 3 


27.3-30.2 


28.66 


1.343 


29.6-33.7 


32.02 


0.889 


T 4 


32.0-36.5 


34.09 


1.880 


42.0-56.5 


48.69 


3.098 


T 5 


27.2-28.6 


27.97 


0.506 


31.6-37.5 


34.01 


1.447 


T 6 


27.4-30.7 


28.91 


1.198 


34.6-39.3 


37.51 


1.138 


Mi 


40-50 


44.6 


4.23 


25-78 


45.9 


15.17 


M 2 


50-80 


70.6 


13.02 


55-82 


70.3 


6.37 


M 3 


69-74 


72.0 


2.14 


49-84 


65.9 


7.55 



Values for temperatures and soil moisture near I'oinacca urceus aestivating in 
the savannah are given in Table I for the reference points of Figure 3. Measure- 
ments were taken on seven snails (mean shell length, 116.7 mm 9.01 S.D.) during 
the morning and on nine snails (mean shell length, 105.3 mm 10.77 S.D.) dur- 
ing the afternoon. Adult snails normally aestivate with a small patch of shell 
surface exposed (Fig. 3), and the hard-baked ground has small cracks which ap- 



310 BURKY, PACHECO, AND PEREYRA 

patently form minute air passages to the brood chamber beneath the shell aperture. 
Figure 3 shows the position of an egg clutch at the beginning of the dry season. At 
the time of these field measurements (end of dry season), only spat were found 
beneath females. Soil moisture content increases with depth. However, in the 
afternoon the soil moisture content immediately below the snails (about 10 cm 
depth) is somewhat less than at 6 cm. This lower moisture content would suggest 
evaporative water loss from the soil adjacent to the snails; however, these dif- 
ferences are not significant (Table I). This does not mean that evaporative cooling 
by the soil is not important. It was not possible to take soil moisture readings at 
10 cm depth for comparison with the level immediately below the snails. Also, 
a certain amount of variation is implicit in these field studies since snails were found 

TABLE II 

Oxygen consumption for active and aestivating snails at 30 'C 



Active 


Aestivating 


Shell length (mm) 
Mean S.D. 




110. 


2 





10 


.64 


104 


.7 





12 


.59 


Live (body & shell) weight (g) 
Mean S.D. 


294. 


5 





104 


.18 


169 


.6 





64 


.34 


Body wet weight (g) 
Mean S.D. 




101. 








36 


.37 


58 


.6 





23 


.58 


Body dry weight (g) 
Mean S.D. 




20. 


7 





12 


.81 


12 


.7 





4 


.42 


nil Oo/hr/mean snail 95% confidence limits 


5. 


07 





1 


.458 


1 


.05 








.439 


M l O 2 /hr/g wet 95% 


confidence limits 


54. 


57 





15 


.289 


20 


.82 





12 


.452 


Ml O 2 /hr/g dry 95% 


confidence limits 


310. 


72 





73.551 


91, 


81 





49. 


902 



aestivating at varying distances from areas of standing water, and these measure- 
ments were taken over periods of one hour 15 minutes and four hours 15 minutes 
during the morning and afternoon, respectively. At all times the temperature of 
exposed shell (T 4 ) was higher than body core temperature (T 6 ) due to the ab- 
sorption of solar heat. The difference between T 4 and T 6 indicates transfer of heat 
from the shell surface to the body tissues. In the morning the soil temperature 
beneath snails (T 5 ) is generally less than the soil temperature at 10 cm depth (T 3 ), 
thus indicating evaporative water loss (cooling) of the soil adjacent to the snails. 
In the afternoon the relationship is reversed with T 5 greater than T 3 indicating 
transfer of body heat to the surrounding soil at a time of greatest solar absorption. 
The high shell temperature is a result of the dark brown to black coloration (sig- 
nificance will be discussed). Also, the temperature of exposed shell surface (T 4 ) 
is greater than the air temperature (TJ indicating heat transfer from the exposed 
shell surface to the air. 

The weight loss during aestivation is given in Figure 4. A long term experi- 
ment on 126 snails (mean shell length, 91.5 mm; live weight range, 32.6-288.2 g 
at start) was terminated 526 days later with 83 living snails. A short term ex- 
periment on 41 snails (mean shell length, 100.3 mm; live weight range, 81.5-385.6 g 



TEMPERATURE REGIME IN POMACEA 



311 



100 



o 

cc 

LJ 
M 



90- 



O 

t- 
T 
O 



80-- 



O 

uj 70 + 
O 

< 

h- 
z 

UJ 

U 
cc 
60;; 





I 



J 



! 



100 



200 



300 
DAYS 



400 



FIGURE 4. Weight loss during aestivation of Powacea urceus in the laboratory is plotted 
against time. The mean weights of a long term experiment (closed circles) and a short term 
experiment (open circles) are given with 95% confidence limits. For further details, see text. 



at start) was terminated 137 days later with 32 living snails. The loss of snails dur- 
ing the course of these experiments was due to mortality and the utilization of some 
specimens for other experiments (only one died in the short term experiment). The 
duration of the short term experiment approximates a normal period of aestivation 
(about 4.5 months) with 19% weight loss (about 35% of tissue weight). The long 
term survival (526 days) under these conditions with 34% weight loss (about 62% 
of tissue weight) is remarkable. This represents an aestivation period of about 
four times the normal. The initial weight loss is most rapid and represents the 
laying of eggs by some females and probably the loss of water retained in the 
mantle cavities of both males and females. On occasion, one of us (A. J. B.) 
recorded relative humidities as low as 45% and as high as 80% at the laboratory 
during the dry and rainy seasons, respectively. The weight loss during aestiva- 
tion would give gross values of between 118 and 233 mg H 2 O per day for a snail 
with a tissue weight of 100 g for the long and short term experiments, respectively. 
The oxygen consumption values for 15 aestivating and 18 active P. urceus are 
summarized in Table II. Rates were measured at 30 C since this is within the 
normal range for body temperatures during aestivation (Table I) and during 
activity in the rainy season when the diurnal temperature ranges can be 28.6- 
32.8 C (Burky and Burky, in preparation). The total oxygen consumption for 
active adults is about five times greater than for aestivating snails. Also, the 



312 BURKY, PACHECO, AND PEREYRA 

values in Table II draw attention to the marked differences in body weights for 
active and aestivating snails of similar size. 

Five aestivating adult snails were placed in each of three sealed desiccators with 
dial hygrometers: (1) air and CaCl 2 ; (2} KOH, CaQ 2 , pyrogallol, and re- 
placement of air with N 2 ; (3) same as No. 2 but without KOH. In five days the 
chamber humidities had risen to 32, 44, and 41%, respectively with activity of two 
and three snails in chambers (2) and (3), respectively. On day 26 all snails in 
chambers (2) and (3) were dead. All snails in chamber (1) were living on day 
77. Based on the oxygen consumption rates of Table II, chamber (1) contained 
more than the two liters of air necessary to supply oxygen for these snails. The 
mortality in the other two chambers indicate that Pomacea urceus can not survive 
for prolonged periods under anaerobic conditions. 

DISCUSSION 

As pointed out in the introduction, respiration in ampullariid snails is am- 
phibious. Although many ampullariids are completely aquatic, some are terrestrial 
for oviposition and/or for aestivation during periodic dry seasons (Prashad, 1925, 
1932; Ranjah, 1942; Meenakshi, 1956, 1957, 1964; Coles, 1968; Little, 1968; 
Burky, in preparation ; and others). It follows that temperature regulation for these 
snails is also amphibious. During the wet season, ampullariids are directly subject 
to the temperature fluctuations of the water. Prashad (1925, 1932) reports that 
Pila globosa commonly makes long excursions on land and will remain out of water 
for a few hours to lay eggs, but there is no information on temperature. Apart from 
this, information indicates that Pila globosa (Prashad, 1925, 1932; Ranjah, 1942; 
Saxena, 1955), Pila vircus (Meenakshi, 1956, 1957, 1964), Pila ovata (Coles, 1968, 
1969), Pomacea lineata (Little, 1968), Pomacea depressa (Little, 1968), and 
Pomacea urceus (Pain, 1950; Burky, in preparation, and this report) are in 
aestivation for most of their terrestrial phase. 

This study shows that Pomacea urceus (adults) can regulate body temperature 
under experimental conditions (Figs. 1 and 2) by exposing foot and mantle through 
opercular movements. In pulmonate slugs there are additional problems of water 
loss since there is no shell for protection (Hogben and Kirk, 1944; Dainton, 1954a, 
1954b). At controlled temperatures it is indicated that water evaporation from 
P. urceus may be similar to loss from a free water surface. Significantly, evaporative 
water loss for active Helix aspersa is similar to that of a free water surface (Machin, 
1964, 1965), but water loss from the mantle of inactive H. aspersa can be regulated 
(Machin, 1965, 1966). Water loss for pulmonate snails under arid conditions is 
inadequate for temperature regulation (Pomeroy, 1968; Yom-Tov, 1971a; Schmidt- 
Nielsen et al., 1971). For Helicclla virgata (Pomeroy, 1968) and for Trochoidea 
seetzeni (Yom-Tov, 1971a) the climbing on vegetation is important for regulation 
of body temperature. This is a function of their position in the temperature gradi- 
ent between soil and air. Sphincterochilla boissieri survives exposure to the sun 
on the ground surface of the desert by the high reflectivity of its light colored shell 
(Yom-Tov, 1971a) and by slow conduction of heat from the substrate (Schmidt- 
Nielsen et al., 1971). Under field conditions the dark shell of Pomacea urceus 
absorbs solar radiation and heat is transferred to the air and to the body tissues. 
Heat then flows from the snail to its burrow walls by conduction and subsequently 



TEMPERATURE REGIME IN POMACEA 313 

some heat is dissipated by evaporative water loss from the earth next to the shell 
(Fig. 3). The water loss during aestivation under experimental conditions (118 
233 mg HoO/clay/100 g tissue) would be inadequate to be an important factor 
for sustained temperature regulation in the field. However, the ability to regulate 
temperature by evaporative cooling could act as a supplement to the cooling af- 
forded by the ground. Pouiacca itrccits can survive 14 months with a loss of 62% 
of its tissue weight (35/y during a normal dry season). Pomace a lincata can sur- 
vive for over 13 months with a loss of greater than 50% of its tissue weight 
(Little, 1968). Normally, Pila virens loses 5% of its tissue water over a six month 
period, but under experimental conditions (operculum removed) snails die when 
50% of their tissue water has been lost (Meenakshi, 1964). Aestivation in Pila 
firms is obviously different since it is found deep in the ground with the operculum 
and dried mucus sealing the shell opening (Meenakshi, 1964). Meenakshi (1964) 
also showed that Pila vircns does not survive at or above 40 C. nor at or below 
20 C. The present data on Pouiacca iirccus suggests an upper lethal temperature 
between 40 and 45 C with temperature regulation generally below 41 C (Table I ; 
Figs. 1 and 3). A lower lethal temperature was not determined but it would not be 
surprising if it is similar to that found for Pila- I'ircns since the data of Burky and 
Burky (in preparation) and Table I indicate that field temperatures for Pouiacca 
urccns are normally above 24-25 C. 

Excretion must also be considered in water balance. It is known that Pila 
ovata (Visser, 1965), Pila globosa (Saxena, 1955; Raghupathiramireddy and 
Swami. 1963), Pouiacca lincata (Little, 1968), and I'ouiacca urccns (Pacheco and 
Pereyra. unpublished data) all excrete uric acid during aestivation. For aestivating 
I'ouiacca- urccns 4.2. 4.4, and 8.1 mg uric acid per gram of fresh tissue are found 
for hepatopancreas, foot, and kidney, respectively (Pacheco and Pereyra, unpub- 
lished data). The water saving advantage of uricotillism during aestivation is 
apparent. 

The exposure of a small patch of the brown to black shell is a seemingly non- 
adaptive condition. It is known that many snails of deserts have light-colored 
shells (Morton, 1958; Russell Hunter, 1964; Pomeroy, 1968; Yom-Tov. 1971a; 
Schmidt-Nielsen et al., 1971) and that this coloring acts to reflect solar energy 
(Yom-Tov, 1971a). Exposure of the shell by Pouiacca urccns appears to promote 
the formation of cracks about the animal as the ground surface dries. These cracks 
undoubtedly aid in evaporative water loss from the area of the shell and for the 
diffusion of air (metabolism is discussed below). The dark sculptured shell has 
a cryptic function since it blends with the ground surface, i.e., visual protection 
from predation by birds. 

Oviposition in Pouiacca itrcetts is at the beginning of the dry season after the 
female has burrowed into the surface mud. Clutches are laid beneath the shell 
aperture where the spat hatch and aestivate until the rains start about four months 
later. Camouflage and temperature regulation is apparently important for the 
protection of spat since they are unable to survive exposure to the tropical sun 
under experimental conditions (this report) or in the field during the dry season 
(Burky, in preparation). It is also known that adults reach a shell length of at 
least 85 mm before going into aestivation at the beginning of the dry season (Burky, 
in preparation). This suggests that there may be a minimum body volume for 
maintenance of body temperature and for water balance during the dry season. 



3U BURKY, PACHECO, AND PEREYRA 

Although aestivation in /'//</ virens is anaerobic (Meenakshi, 1956, 1957, 1964), 
it is aerobic in Pila ovata (Visser, 1965; Coles, 1968, 1969) and in Pomacca urceus. 
Further, Pila virens accumulates lactic acid during aestivation (Meenakshi, 1956, 
1957) while Pila ovata (Coles, 1968) and Pomacca urceus (0.219 and 0.183 ing 
lactic acid per gram of fresh tissue for foot and hepatopancreas, respectively ; 
Pacheco and Percy ra. unpublished data) do not accumulate lactic acid during 
aestivation. The special problems of animals under anaerobic conditions have been 
discussed (von Brand, 1946; Dales, 1956; Beadle, 1961; and others). Both 
Pomacca urceus and Pila ovata (Coles, 1968) have a similar rate of oxygen con- 
sumption with the rate during aestivation being one fifth and one sixth that for 
active snails respectively. In Pila ovata, aestivating snails elevate oxygen con- 
sumption when disturbed (Coles, 1968). This type of disturbance reaction is 
undoubtedly true for Pomacca urceus since aestivating adults have been observed 
(by A. J. B.) to extend their body when the shell is knocked. Pomacca urceus 
aestivates next to the ground surface where conditions in the dry soil are probably 
not anaerobic. The soil cracks around the shell make oxygen more available to the 
snail. Further, a good oxygen supply is assumed necessary for the developing 
eggs in the brood chamber beneath the female. In his discussion of anaerobic 
habitats, von Brand (1 ( H6) points out that in most soils the oxygen concentration 
is sufficient lor aerobic respiration, llowever. after rain when the soil is wet, the 
exchange of gases with the air is reduced and the ground oxygen can be rapidly 
depleted. Anaerobic conditions for aestivating Pomacca urceus are most likely to 
occur at the beginning and at the end of the dry season. It would not be sur- 
prising if these snails use pulmonary respiration via their siphon when they first 
burrow into the mud at the water's edge. At the end of the dry season the first 
heavy rains could fill the burrows and cut off the oxygen supply, but burrows are 
superficial and anaerobic conditions could involve only a few hours. Also, these 
snails become active in the presence of water at the end of the dry season. This 
is particularly rapid in spat since only minutes are necessary for full activity. 

Attention has been drawn to the animal sequence of rainy and dry seasons 
of the Venezuelan plains and to the terrestrial and aquatic phases in the life cycle 
of these snails. The ampullariid gastropods are doubly adapted in terms of their 
"amphibious"' respiratory structures. The presence of unlimited water or its rela- 
tive absence provides different conditions for temperature experience and activity. 
The behavioral adaptation of burrowing and the subsequent inactivity (aestivation 
with lowered metabolism) are of advantage for survival during the dry season. 
Further, uricotellism and tolerance of dehydration are both adaptive under arid 
conditions. The existence of wet and dry seasons stress the importance of adapta- 
tions for an amphibious way of life. 



We would like to thank Dr. T. Pain for having examined specimens of this study 
and for having identified these snails as typical Pomacca urceus (Miiller) ; Pro- 
fessor Rafael Martinez for making equipment available and for informative dis- 
cussions about the natural history of snails in the Llanos region of Venezuela ; 
Eduardo Miranda and Oswaldo Travieso for their assistance in collecting snails ; 
the other students who aided in the recording of data ; and Kathleen A. Burky 
for assistance in field collecting and during the preparation of this paper. 



TEMPERATURE REGIME IX POMACEA 315 

SUMMARY 

1. It has been demonstrated that adult snails generally regulate their body 
temperature below 41 C under experimental conditions and that their upper 
lethal temperature is between 40 and 45 C. 

2. Field data indicate that under natural conditions adult body heat is trans- 
feree! to the ground of the aestivation burrow by conduction and that this heat is 
at least in part dissipated by evaporative loss of soil moisture. 

3. Under experimental conditions snails can survive for an aestivation period 
of four times the normal length and with a loss of 62% of their tissue weight. This 
level of experimental water loss would be inadequate as the only agent of tempera- 
ture regulation under field conditions but could be a supplement to heat transfer 
to both soil and air as well as evaporative cooling afforded by the ground. 

4. Aestivating adults can survive many days of direct exposure to the tropical 
sun ( out of burrow ) while juveniles are dead within two hours or less. 

5. The metabolism of aestivation is aerobic with oxygen consumption about 
one fifth that of active snails. 

6. Females provide protection from high temperatures and from water loss for 
eggs and spat during the dry season. The adaptiveness of superficial aestivation 
burrows is discussed in relation to the needs of aerobic metabolism for adults and 
developing eggs. 

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MACHIN, J., 1964. The evaporation of water from Ilcli.r aspcrsa I. The nature of the evapo- 
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Reference : Biol. Bull., 143 : 317-331. (October, 1972 ) 



REGENERATION OF THE PROBOSCIS OF MURICID GASTROPODS 

AFTER AMPUTATION, WITH EMPHASIS ON THE 

RADULA AND CARTILAGES 

MELBOURNE R. CARRIKER, PHILIP PERSON, RICHARD LIBBIN. 

AXD DIRK VAN ZANDT 

Systcmatics-Ecology Program, Marine Biological Laboratory, H'oods Hole. Massachusetts 

1-543; Special Research Laboratory for Oral Tissue Metabolism, I'eterans Administration 

Hospital. Brooklyn. New York 11209; and Department of Bioloi/y. I'ard Collciie. 

. \nnandale-on-l I ndson, Ne^v York 12504 

Boring of prey shells by muricid gastropods involves the close interaction of the 
proboscis, propodium, and the accessory boring organ (ABO) in a predictable cycle 
which repeats itself continuously throughout the process of boring of each borehole. 
Although the radula serves only a minor part in shaping the borehole, it appears that 
both the radula and the ABO are necessary to effect normal penetration (Carriker 
and Van Zandt, 1972). \Yhether this is so could be tested by inactivation of one 
and then the other organ in different individuals and noting the effect on penetration. 
The first objective of the present study was to examine the effect of removal of the 
proboscis on boring. 

In 1957 an adult Urosalpin.r cincrca in our laboratory caught its proboscis be- 
tween the glass and shell of an oyster model ( Carriker and Van Zandt, 1972), and 
after tugging for some time to free itself, tore away the proboscis leaving the distal 
third wedged in the model. The snail recovered and resumed boring of prey in 
20 days. The previous year Demoran and Gunter (1956) experimentally ampu- 
tated the distal portion of the proboscis of several thaisid boring snails, Thais 
ha-eiiuistoma, and reported that these gastropods regenerated the proboscis within 
three weeks provided the organ had been cut off cleanly. From time to time the 
senior author has dissected large adult Urosalpin.v cincrca collected in the field in 
which the anterior portion of the proboscis, though abnormally small, externally 
appeared morphologically normal. The small size of the proboscis tip indicated that 
these snails had accidently lost and regenerated the proboscis. These several ob- 
servations suggest that regeneration of the complex buccal mass in Urosalpin.v 
cincrca (Carriker, 1943) and other predatory marine gastropods may occur nor- 
mallv in nature. The second objective of this investigation was thus to determine 
how commonly and at what rate regeneration occurs in the laboratory after experi- 
mental proboscisectomy. A preliminary summary of the results of these studies 
was published by Carriker (1959, 1961). The radula, being a relatively hard struc- 
ture (Carriker and Van Zandt, 1972) provides a readily measurable parameter for 
quantitative determination of regeneration. 

During the past several decades regeneration of cartilages has been investigated 
almost exclusively in vertebrate animals. Recently, however, following a long 
period of neglect, invertebrate cartilages again came under serious study (Person 
and Philpott. 1969; Mathews, 1968). As part of a survey of the nature and prop- 

317 



3 IN CARR1KER, PERSON, LIBBIX, AXD VAX ZAXDT 

erties of cartilages in various invertebrate phyla, Person became interested in their 
regenerative capacity. In the reports by Demoran and Gunter ( 1956) and Carriker 
(1961) neither specific mention nor details were given concerning regeneration of 
the odontophoral cartilages which were removed by proboscisectomy. The third 
objective of the investigation was therefore to ascertain whether complete histologi- 
cal regeneration of these structures occurs following proboscisectomy. 

[MATERIALS AND METHODS 

Healthy well fed individuals of I 'rosa/pin.v cincrca (Say), Urosalf>in.v cincrea 
jollyensis Baker, and linplcnra caudata cttcrac Baker (Family Aluricidae, Class 
Prosobranchia) were employed in the investigation. Three separate experiments 
were run. The first was carried out during the summer of 1958 at the University 
of Nortli Carolina Institute of Fisheries Research, Morehead City, North Carolina, 
and employed Urosal[>i)i.\- cincrca from the local sounds of North Carolina, and 
Eu pleura candata cttcrac from Chincoteague Bay, Virginia. The second (1965) 
and third (1970) experiments were conducted at the Marine Biological Laboratory 
and employed only Urosalpinx cincrca follyensis from Wachapreague, Virginia. 

Snails were shipped airmail and maintained in the laboratory in rapidly running 
seawater. They were fed oysters and acclimatized readily to laboratory conditions. 
At the Institute of Fisheries Research the temperature of the running seawater 
during the experiment ranged between 23.5 and 33.0 C, and the salinity between 
27 and 32^ f . At the Marine Biological Laboratory in 1965 the temperature of the 
running seawater varied between 18.0 and 22.5 C, the salinity between 31.9 and 
32.0% and the pH between 8.09 and 8.11. In 1970 the temperature fluctuated be- 
tween 20.3 and 23.8 C. the salinity between 31.3 and 31.5V and the pH between 
8.03 and 8.14. During the daytime snails were illuminated by daylight coming 
through the laboratory windows, and during the early part of the evening by stan- 
dard overhead artificial laboratory light. 

The technique devised for proboscisectomy was developed in the summer of 1957, 
and was as follows : Into a tray of running seawater we placed a kitchen-type plastic 
dish, approximately 7.5 by 7.5 by 10 cm, whose sides had been perforated many 
times to allow ready passage of seawater. A plastic screen, pore size approximately 
1 mm, had been cemented into the dish to provide an upright diagonal divider, 
creating two triangular compartments. In the upstream compartment we placed a 
freshly shucked live oyster with its flesh against the screen ; in the downstream 
compartment we placed four to six snails. After several hours one or more snails 
were attracted to the oyster, and after mounting the screen and inserting their 
proboscides through the screen, they began to feed on the oyster. After the snails 
had been feeding for a few hours, they could be pulled gently away from the screen 
without detaching the everted proboscis from the oyster, thereby extending the 
proboscis further. The considerable length of the proboscis when fully everted, 
about the same as the height of the snail shell, facilitated the operation. An iris 
scissors was then carefully inserted between the screen and the snail, and the pro- 
boscis was cut quickly and cleanly close to its base, removing with it the buccal mass 
and more or less of the radular sac depending on the plane of amputation ( Fig. 1 ) 
and the degree of extension of the sac. The snails were then allowed to recuperate 
undisturbed in running seawater. Excision of the buccal mass, radula. supporting 



REGENERATION OF GASTROPOD PROBOSCIS 



319 



odontophoral cartilages, and other structures of the proboscis were verified by exam- 
ination of the amputated proboscis with a dissecting microscope. For the details of 
normal morphology of the adult proboscis, see Carriker (1943). Xo mortality re- 
sulted from the proboscisectomies, and all snails recovered rapidly. 

The 1958 experiment was designed to determine (a) the number of days after 
proboscisectomy when boring would resume, and (b) the rate of anatomical regen- 
eration of the proboscis after proboscisectomy. A total of 64 snails was successfully 
proboscisectomized, 32 snails for (a) and 32 for (b) (see below). Each of (a) 
and (b) included 16 Urosalpiu.r cinerea (8 males and 8 females, 4 medium and 4 
large individuals of each sex) and 16 Euplenra candata etterae (8 males and 8 
females, 4 medium and 4 large individuals of each sex). The Urosalpm.r cinerea 
ranged in shell height from 16.5 to 25.9 mm, and the Enplcura candata etterae from 
19.6 to 34.2 mm. 



D-e 







FIGURE 1. Diagrams illustrating the gross anatomy, amputation, and regeneration of the 
proboscis of Urosalpin.v cinerea, shell height approximately 25 mm: (a) median plane of the 
proboscis retracted within the proboscis sac in the cephalic hemocoel ; (b) line a, approximate 
plane at which proboscis amputated; (c) blastema joining the proboscis stump and ends of 
esophagus, buccal artery, and accessory salivary gland duct ; (d) regenerated proboscis tip, 
buccal mass, and radular sac ; a, plane of amputation ; b, blastema ; ba, buccal artery ; bm, buccal 
mass; ch, cephalic hemocoel; e, esophagus; gd, accessory salivary gland duct; m, mouth; ps, 
proboscis sac ; rs, radular sac ; scale bar, 1 mm. 

In (a), immediately after proboscisectomy, snails were placed in small trans- 
lucent perforated plastic dishes in running seawater filtered through coarse sand and 
shell fragments, each snail isolated with a live oyster 5-7 cm long and cleaned of 
encrustations. After remaining quietly in a corner of the dish for a day or so, each 
snail crawled about, and in time was attracted to the oyster and mounted it. There- 
after each day with a needle we gently pushed the midanterior portion of the pro- 
podium of the snail back to a point immediately under the ABO. If boring had 
begun, the initial borehole was clearly evident, the snail was sacrificed, and the 



320 



CARRIKER, PERSON, LIBBIN, AND VAN ZANDT 





FIGURE 2. a-e : light micrographs of radulae of L"ro^o//'/H.i' cincrca: (a) anterior end of 
normal radula of adult snail on subradular membrane; scale bar, 35 /JL ; (b) marginal (single 
cusped) and rachidian (tricusped) teeth of normal radula of adult snail; scale bar, 20 /x ; (c) 
parts of regenerated radula 100 days after snail tore off the anterior third of the proboscis which 
was wedged in a laboratory device, snail shell height 25 mm; scale bar, 20^; (d) anterior 
portion of regenerating radula 12 days after proboscisectomy ; scale bar, 20 p ; (e) posterior 
portion of same radula as in (d) ; scale bar, 20 p.. f-g : light micrographs of radulae of Euplcura 



REGENERATION OF GASTROPOD PROBOSCIS 321 

proboscis was examined for extent of regeneration. If boring had not begun, the 
snail and oyster were returned to the dish. Oysters were changed weekly with 
individuals freshly collected in the field. Oysters held in captivity, even in running 
seawater, tend rapidly to lose their attractiveness after a few days (Carriker and 
Van Zandt, 1972), possibly because of decreased food. 

In (b) proboscisectomized snails were likewise placed in dishes in running 
filtered seawater, but without oysters. Eight snails (a large and a small male, and 
a large and a small female of each species) were sacrificed on the 4th, 8th, 12th, and 
16th day after amputation, and the regenerating proboscides were examined for 
degree of anatomical regeneration. 

At the time of sacrifice the shell of the recuperating snail was cracked open by a 
blow with a hammer, and the animal was removed. The regenerating proboscis was 
then excised from the cephalic hemocoel of the snail, opened under a dissecting mi- 
croscope, held in position with minute dissecting pins on the exposed surface of a 
rubber eraser embedded in wax in a dissecting pan, and examined under seawater. 
Tissues were stained with Little's methylene blue as the dissection progressed. 
Radulae, if present, were freed of soft tissue in 10% KOH, washed, stained in \% 
aqueous chromic acid, dehydrated in alcohols, and mounted flat in Euparol to facil- 
itate examination of the teeth. Because of the tendency of radulae to curl, some of 
them turned on the side during mounting. Study and measurement of the teeth was 
done with an ocular micrometer in a compound microscope. 

The 1965 experiment was designed to provide regenerating proboscides at inter- 
vals of 4, 8, 12, and 20 days after proboscisectomy for histological examination. 
Thirteen individuals of Urosalpin.r cincrea follyensis, ranging in shell height from 
25 to 43 mm. were utilized, three specimens each for the 4, 8, and 12 day periods, 
and four for the 20 day period. Regenerating proboscides were everted from the 
cephalic hemocoel (except in the earliest cases where the proboscis was too small 
to permit orientation), excised at the base, fixed in cold Bouin's, dehydrated in ethyl 
alcohols, and embedded in paraffin. Sections were cut 7 /JL thick on a standard 
microtome parallel to the long (anterior-posterior) axis. One half of the sections 
in each series was stained with Lillie's modification of Masson's trichrome stain, and 
alternate serial sections were stained with astra blue, Weigert's iron hematoxylin, or 
Mallory-Heidenhain's stain. 

In the 1970 experiment, designed to study the histology and cytology of the 
regenerating cartilages of the buccal mass, recuperating Urosalpin.r cinerca jollyensis 
(ranging in shell height approximately from 27 to 38 mm) were sacrificed at time 
intervals of 3, 7, 11. and 19 days post-proboscisectomy. A minimum of four snails 
was used in each group. The regenerating proboscides were removed under a dis- 
secting microscope, and half were placed in Bouin's fixative for paraffin embedding, 
and the remainder were frozen immediately on a quick-freeze stage. Frozen sec- 
tions 8 ,u, in thickness were prepared in a Slee freezing microtome for toluidine-blue 
staining at pH 3. The paraffin embedded sections (5^ thick) were stained with 
Wright's nuclear stain, astra blue, or Mallory triple stain. 

caitdata cttcrae; (f) anterior end of normal radula on subradular membrane, snail shell height 
25 mm; scale bar, 23 /tt; (g) regenerating radula 8 days after experimental proboscisectomy, 
snail shell height 28 mm, anterior portion of the radula to the left, posterior portion to the right ; 
scale bar, 23 /j,. 



322 



CARRIKER, PERSON, LIBBIN, AND VAN ZANDT 



RESULTS 
Regenerative changes and rates 

In 1958 all 32 individuals of Urosalpin.v cinerca and Enplenra caudata etterae 
which were allowed to resume boring (experiment a), had regenerated the pro- 
boscis, and this organ, though proportionately small, appeared normal under the 
dissecting microscope. Traces of pink color (probably myoglobin) appeared in the 
musculature of the buccal mass by the 15th day, and by the 20th day this became 
more intense. The normal buccal mass in the adult snail is brightly colored. The 
32 individuals of both species which were sacrificed at four day intervals after 
proboscisectomy (experiment b), had all begun regeneration of the proboscis, the 
degree of development increasing with time past amputation. By the 4th day, at 
least a filmy cap of loose tissue covered the stump of the proboscis and bound the 
amputated ends of the esophagus, buccal artery, ducts of the salivary glands, and 
other tissues to it (Fig. Ic). In two snails (one of each species) the external form 
of the minute radular sac was already clearly evident. Between the 8th and 12th 
days after the operation, the four individuals of both species had formed minute 
normal proboscides and radulae (Fig. Id), and by the 16th day the radulae had 
approximately doubled in length. 



35 - 



30 



O 



RESUMED 
u BORING 

9 
c? o 



UJ 

c/2 
O 

in 
o o 

OD ^ u 

O 

LT_ 
Q_ 

g '5 



< ' 
Q 



NON 
BORING 




Urosalpinx 







Eupleura 



12345 12345 

RADULAR LENGTH, mm 

FIGURE 3. Extent of regeneration of radulae of male and female Urosalpin.v cinerca and 
Eupleura caudata etterae 4, 8, 12, and 16 days after proboscisectomy ("non boring"), and at 
resumption of boring after proboscisectomy ("resumed boring"). 



REGENERATION OF GASTROPOD PROBOSCIS 



323 



In two snails, where amputation of the proboscis had taken place at the level of 
the radular sac, a part of the large original radula, now devoid of its sac but other- 
wise intact and showing no sign of dissolution, was found in the base of the re- 
generating proboscis. 

In the 8th and 12th day regenerating radulae, the earliest rachidian and marginal 
teeth were small and slightly misshapen ( Fig. 2 ) . In one radula the early mid- 
rachidian cusp was forked for several initial transverse rows, then in one row 
changed from the forked to the normal unicuspid condition. Earliest rachidial 
teeth were separated from each other more than were normal teeth, and even in very 
small radulae were heavily worn by abrasion, suggesting that the snail starts rasping 
soon after the teeth are first formed. Teeth increased rapidly in size along the long 
axis of the radula with time, and successive rows achieved the normal form quickly, 
the rachidian teeth first, and some time later, the marginal teeth. Earliest regener- 
ating marginal teeth started as short, weak, thread-like structures. 



35 



25 



CO 20 



15 



Urosalpinx 



9 - 

(f - O 



L 



J 



_L 



Eupleura 






_L 



1 



012345012345 
RADULA LENGTH WHEN BORING RESUMED, MM 

FIGURE 4. Length of radulae and shell height of male and female Urosalpinx cinerca 
and Eiiplcura caitdata cttcrac at resumption of boring. 



The approximate rate of regeneration of radulae in Urosalpinx cinerca and 
En pleura candata cttcrae is plotted in Figure 3. Initial regeneration was a little 
faster in Eupleura can-data ctterac than in Urosalpinx cinerea, but the length of the 
radula increased at a faster rate in some Urosalpinx cinerca. There were noticeable 
differences in the growth rate of radulae among individual snails, but no consistent 
differences between the growth rate of radulae of males and females. 

Although the rate of increase of radulae of both species was surprisingly rapid 
and relatively uniform, the time of onset of shell penetration of prey varied from 11 
to 34 days (Fig. 3). No obvious differences in the onset of boring by males and 
females was evident. Likewise there was no association between the length of the 
radula and the height of the snail shell when boring was resumed (Fig. 4). 

Measurement with an ocular micrometer of the widest portion of the base of 



324 



CARRIKER, PERSON. LIBBIN, AND VAN ZANDT 



rachidian teeth at the anterior (oldest) and posterior (newest) ends of (a) normal 
radulae, ( b ) regenerating radulae of snails allowed to resume boring after probosci- 
sectomy, and (c) regenerating radulae of snails sacrificed at intervals after amputa- 
tion, clearly demonstrated the rapid rate of enlargement of teeth with time after 
proboscisectomy (Fig. 5). However, there was noticeable variation in size between 
old and new rachidian teeth in some of the normal adult snails of both species : in 
some, newest teeth were smaller than old ones ; in others, they were larger ; and in 
still others, the radnla was constant in size throughout its length. Rate of widening 
of rachidian teeth was maximal in the earliest stages of regeneration of the proboscis 
(Fig. 5). 

Examination of incomplete boreholes excavated by snails which were allowed to 
bore after regeneration of the amputated proboscis, disclosed that 12 of the holes 
made by Vrosalpinx cincrca, and 13 by Eu pleura candata ctterea, were normal as 
to size and shape (see Carriker and Yochelson, 1968), and 4 and 3, respectively, 
were slightly abnormal only in shape. The abnormality, however, appeared to be 
caused more by the uneveness and irregularity of the growth rings in the oyster 
shell than by malfunctioning of the radula. 



100 



i 
~ 



- 



\ 

Q 

r 



I 

I 
-J 



75 




Urosalpinx 



CONTROLS 

- RESUMED BORING 

- NON BORING 



Eupleura 



_L 



60 120 180 240 300 360 I 60 120 180 

NUMBER TRANSVERSE ROWS RADULAR TEETH 



240 



300 



360 



FIGURE 5. Change in width of the base of rachidian teeth of Urosalphi.r cincrca and 
Eupleura candata cttcrac from the anterior (old portion) to the posterior (new portion) of the 
radulae: in normal snails ("controls"), in snails whose proboscides were amputated and then 
allowed to regenerate and resume boring ("resumed boring"), and in snails whose proboscides 
were amputated and then the snails were sacrificed at 4, 8, 12, and 16 days after amputation 
("non boring"). Numbers beside points refer to the number of days after proboscisectomy when 
snails were sacrificed. Teeth were counted from the anterior to the posterior of the radulae. 



REGENERATION OF GASTROPOD PROBOSCIS 



325 




bm 



FIGURE 6. Representative stages in the regeneration of the proboscis ot' I'rasiilpin.r cincrcn 
follyensis following proboscisectomy. Snails were sacrificed 4, 8, 12, and 20 days after amputa- 
tion ; light micrographs ; Astra blue, Weigert's iron hematoxylin, and Mallory-Heidenhain's 
stains: (a) initial stage illustrating early blastema, 4 days after amputation; scale bar, 40 /x ; 
(b) early stage, 12 days after amputation; scale bar, 85^; (c) intermediate stage showing radula 
and radular sac, 8 days after amputation; scale bar, 100 /j. ; (d) new proboscis, withdrawn in 
proboscis sac, 20 days after amputation; scale bar, 100 n ; (e) new proboscis, everted, 20 days 
after amputation; scale bar, 100 ,u ; be, buccal cavity; b, blastema; c, cap cell; bm, buccal 
musculature ; ct, cartilage, e, esophagus, m, mouth ; o, odontophore ; od, odontophoral muscu- 
lature ; oi, old proboscis integument ; ph, proboscis hemocoel ; ps, proboscis sac ; ri, regenerating 
proboscis integument; rs, radular sac; r, radula, sd, salivary gland duct. 



326 



CARRIKER, PERSON, LIBBIN, AND VAN ZANDT 



on ^~ -/ ;..>?* ' ':- : .; 

<i : .. V- 



* ' " f ' .-V ,* * fr. " * / 

': ct ' 



IP^ 

,.,i. '".*...^ / 

c ,v " t,.e..- , *.. 



C^cr..-T . - . ' ?; ^*^.^^S 
8 *' t^ r ' ' Vf ' '^^5^ 

^Pt ^'.-j-vr.<rt . ^ .-Sa 



*/." ;. .-cV*^ 

^ / " /vi^-ir^V^C 

" % -. ; .;^r , ,H*>?. \-&< 



'- ^ v 

; *r z t^i 

.Vy V I 







FIGURE 7. Representative stages in normal and regenerating odontophores of Urosalpin.v 
cincrea follycnsis: (a) frontal section of odontophoral cartilages in nonoperated (control) snail : 
frozen section, toluidine blue stain; scale bar, 85 jj. ; (b) frontal section of regenerating odonto- 
phoral cartilages in a snail 11 days after proboscisectomy, the section is deeper into the cartilage 
than in fa), frozen section, toluidine blue stain; scale bar, 85 M ; (c) frontal section of anterior 



REGENERATION OF GASTROPOD PROBOSCIS 327 

Histology of regenerating proboscides 

Although all 13 proboscisectomized individuals of Urosalpinx cinerea follycnsis 
in the 1965 experiment commenced regeneration of the proboscis tip, the rate of 
regeneration within each time interval varied noticeably, in contrast to the relatively 
uniform results obtained in the 1958 experiment. Whether this was due to sub- 
specific differences, or to variation in the level of amputation of the proboscis, is 
not known. 

General trends in regeneration of the organs of the proboscis of Urosalpinx 
cinerea follyensis are illustrated in Figure 6. Initially a loose mass of cells, includ- 
ing numerous amebocytes, formed over the cut end of the proboscis and joined this 
to the cut ends of the esophagus, buccal artery, and other tissues (Fig. 6a). The 
epithelium and muscular layers of the esophagus then grew forward into the 
blastema, and the integument of the proboscis extended over the blastema as a thin 
epidermis one cell thick with mucous cells (Fig. 6b). Simultaneously muscle fibers 
appeared within the blastema. From this mass arose the integument and muscula- 
ture of the new proboscis tip, the buccal mass and its musculature, the forward end 
of the esophagus, salivary and accessory salivary gland ducts, arteries, and nerves, 
the odontophore, cartilages, radular sac, radula, and cuticular lining of the buccal 
cavity (Figs. 6c-e, 7d). Odontophoral cartilages, radular sac, and radula were 
histologically distinguishable by the 8th day after proboscisectomy. 

Cytology of regenerating odontophoral cartilages 

In the 1970 experiment regenerating radulae and odontophoral cartilages of 
Urosalpinx cinerea jollyensis were well advanced by the 7th and llth days, respec- 
tively, after proboscisectomy. 

For reference purposes, we will first illustrate the histology of the odontophore 
of a normal nonoperated Urosalpinx cinerea follyensis (Fig. 7a). The cartilages 
were sectioned in a dorsal peripheral plane of the odontophore, and lie surrounded 
by the odontophoral musculature (om). The radular sac (rs) emerges posteriorly 
between the cartilages, and rachidian teeth (rt) are visible on the anterior tip of the 
odontophore in the buccal cavity (be). At its anterior end each cartilage possesses 
a cap of cells (c) which as will be seen, is of complex morphology, and appears 
closely related to tissues of both the odontophoral cartilages and the surrounding 
muscle. Nuclei of the cartilage cells stained orthochromatically (blue), whereas 
the cytoplasm and intercellular matrix of these cells were primarily (but not 
entirely) strongly metachromatic (pink, purple). Muscle fibers, their nuclei, and 

ends of regenerating odontophoral cartilages in a snail 11 days after proboscisectomy. Note 
details of cap cells and both muscle and cartilage cells issuing from them; frozen section, 
toluidine blue stain; scale bar, 35 fj. ; (d) sagittal section through one of the odontophoral 
cartilages in a snail 20 days after proboscisectomy, illustrating the relationship between the cap, 
muscle, and cartilage cells ; Bouin's fixative, Wright astra blue, and Mallory's stains ; scale bar, 
85 ju ; (e) sagittal section through anterior tip of odontophoral cartilage in a snail 20 days after 
proboscisectomy, serial section from same specimen as in (d) ; Bouin's fixative, astra blue, 
Weigert's iron hematoxylin, and Mallory-Heidenhain's stains ; scale bar, 10 /j. ; be. buccal cavity, 
bm, buccal musculature ; c, cap cells ; ct, cartilage ; e, esophagus ; ms, muscle cells ; mt, marginal 
teeth ; om, odontophoral musculature ; r, radula ; rm, radular membrane ; rs, radular sac ; rt, 
rachidian teeth ; sd, salivary gland duct ; sm, subradular membrane. 



328 CARRIKER, PERSON, LIBBIN, AND VAN ZANDT 

the nuclei of the cap cells \vere also orthochromatic, while the cytoplasm and inter- 
cellular substance of the cap cells likewise exhibited metachromasia. 

Regeneration of the cartilages will be described from representative sections of 
the proboscis from snails 11, 19, and 20 days after proboscisectomy. Figure 7b is 
a photomicrograph of a frontal section of the odontophore of an 11 day post- 
proboscisectomized snail. The plane of the section was deeper into the cartilages 
than was the case in the specimen shown in Figure 7a. It is evident that by the 
llth day considerable regeneration of cartilage, radula, and associated tissues had 
taken place. The distribution of metachromasia and orthochromasia in the tissues 
appeared similar to that described for the section of Figure 7a, but the meta- 
chromasia was more intense. Toward the anterior end of each cartilage in Figure 
7b, the cells become smaller in size, and eventually merge with still smaller epitheloid 
cells which form the cap referred to earlier in the nonoperated snail. At the periph- 
ery of the cap, muscle fibers (ms) are in close proximity and appear to interweave 
with the cap cells (c). This is more clearly illustrated in Figure 7c. a higher 
magnification of the cap region in a sx-rial section twice removed from that shown 
in Figure 7b. Both cartilage cells (ct) and muscle cells (ins) blend imperceptibly 
with the cap cells (Fig. 7c), giving the impression of a blastema-like structure 
reminiscent of that seen in vertebrate limb regeneration. This impression was 
strengthened by Figure 7d and 7c which illustrate sagittal sections of regenerated 
odontophores 20 days after proboscisectomy. At low magnification (Fig. 7d ) the 
cap appears as a tightly packed, rapidly dividing mass of cells from which both 
cartilage and muscle are forming. At a higher magnification (Fig. 7e) in a serial 
section tw : ice removed from that shown in Figure 7d, the imperceptible blending of 
both cartilage and muscle cells with those of the cap is unmistakable, and the re- 
semblance to a vertebrate blastema is reinforced. 

DISCUSSION 

The ability of muricid gastropods to penetrate the shell of prey allows them, 
protected by their own shell and for a time by the valves of the prey, to feed on 
otherwise generally inaccessible organisms often much larger than themselves. After 
penetration of the shell and while feeding on gaping moribund oysters, however, 
snails risk loss of the proboscis in t\vo ways: (a) amputation by small crabs and 
fish when the proboscis is extended into the mantle cavity through the borehole, 
and (b) pinching and subsequent loss while the proboscis is inserted between the 
valves. Amputation by both means may occur in nature, though how frequently is 
not known. Valvular motion of normal live prey inhibits boring between the 
edges of the valves ( Carriker and Van Zandt. 1972), so the danger in (b) is 
from a prey which after gaping widely for a time suddenly clamps shut, irritated 
by scavengers feeding on its tissues. 

The present studies demonstrated that regeneration of the proboscis takes place 
in a remarkably short time, and proboscisectomized snails, even the occasional ones 
in which the amputation is ragged, recover. Rapid functional replacement of the 
feeding organ insures survival, and an accident which otherwise might have had 
disastrous consequences, is only a passing inconvenience. The unusual capacity of 
mollusks to regenerate lost parts has been known for a long time CHynian. 1967), 
but the rapid regeneration of so complicated an organ system as the prosobranch 



REGENERATION OF GASTROPOD PROBOSCIS 329 

proboscis has not been reported prior to this investigation and the paper by 
Demoran and Gunter (1956). Isarankura and Runham (1968), by marking the 
radulae of live pulmonates and prosobranchs (including the muricid, Thais lapillus} 
by various techniques, determined that the rate of replacement (forward movement) 
of the radula over the odontophore is continuous. The present studies support 
their findings. 

The effect of removal of the proboscis on the capacity of boring snails to pene- 
trate the shell of oysters was demonstrated by individuals of Urosalpinx and 
Eupleura which were allowed to resume boring after proboscisectomy. In every 
case, boring was initiated only after the radula and associated structures had de- 
veloped normally, anatomically and histologically. Earliest regenerating teeth in- 
creased most rapidly in size. Isarankura and Runham (1968) also reported a very 
rapid rate of replacement of the radula in newly hatched prosobranchs and pul- 
monates, followed by a steady decrease in replacement rate. Although the rate 
of increase in length of radulae of Urosalpinx and Eupleura soon after probosci- 
sectomy was rapid and unform, the time of onset of boring of prey ranged over a 
period of 23 days in different individuals. Thus development of a given length 
of radula did not trigger penetration, and the initiation of boring was stimulated by 
other factors, possibly attractiveness of prey, or the physiological and behavioral 
condition of the snails, or both. The noticeable variation in size between old and 
new rachidian teeth in some adult, nonoperated individuals of both Urosalpinx and 
Eupleura was unexpected. Changes, when they occurred, were gradual down 
the length of the radula, so it is difficult to ascribe them to nutritional causes. 
Resumption of boring only after the radula and associated structures appeared 
anatomically normal suggests that the redula is an essential component of the 
mechanism of shell penetration. Furthermore, the capacity of adult snails with 
small newly regenerating radulae to excavate boreholes of a shape and size similar 
to those of normal adult snails is further evidence that the shape and size of the 
borehole are the products primarily of chemical activity of the accessory boring 
organ rather than of the radula (Carriker and Van Zandt, 1972). 

Regenerating odontophoral cartilages of Urosalpinx are strikingly similar in 
histological appearance to the regenerating limbs of vertebrates, as seen, for example, 
in the salamander (Butler, 1933; Kiortsis and Trampush, 1965; Thornton, 1968). 
In both instances the regenerative process is considerably dependent upon a unique 
cell aggregate, the blastema of vertebrates, and its analogue, which we have called 
the cap cells, of Urosalpinx. Although little is known of the chemistry of the 
cartilage of these snails, its strong metachromatic staining with toluidine blue at pH 
3 indicates the probable presence in the tissue of macromolecular anionic poly- 
saccharides. In Bnsycon, a genus of predatory marine snails, Lash and White- 
house (1960) reported the presence in the odontophoral cartilage of a nonaminated 
polyglucose sulfate. Person and Philpott (1963) have also shown that collagen 
is present in the odontophoral cartilage of Busy con. Although at the present time 
no chemical or ultrastructural data dealing with the odontophoral cartilage of 
Urosalpinx are available, it is likely that some form of anionic polysaccharide and 
also collagen will be found in its tissue. In view of these findings, and of the rela- 
tively rapid regeneration of muricid odontophoral cartilage, we suggest that these 
and other gastropod families may prove useful for the study of cartilage and 
skeletal regeneration. 



330 CARRIKER, PERSON, LIBBIN, AND VAN ZANDT 

The boring habit and the capacity for rapid regeneration of the proboscis pro- 
vide unusual advantages in procurement of food, and these perhaps account in 
large part for the biological success of such muricid species as Urosalpinx cinerea 
and Eupleura can data and their significance as major predators of commercial 
oysters. 



John W. Blake assisted in the investigation in 1958, Barry Martin in 1965, and 
Robert Lipson in 1970. Photographs resulting from the 1958 and 1965 studies 
were taken by Peter J. Oldham. The live specimens of Eupleura caudata etterae 
used in 1958 were supplied by Michael Castagna, Thomas Carter, and George 
Griffith from Chincoteague Bay, Maryland-Virginia ; the live specimens of Uro- 
salpinx cinerea follyensis employed in 1965 and 1970 were supplied by Michael 
Castagna from Wachapreague, Virginia. 

The research in 1958 was supported in part by a grant from the U. S. Fish and 
Wildlife Service; that in 1965 by Public Health Service Research Grant DE 01870 
from the National Institute of Health; and that in 1970 jointly by Public Health 
Service Research Grant DE 01870 and the Veterans Administration. 

Acknowledgment is gratefully made for the many courtesies and generous as- 
sistance which made this study possible. Systematics-Ecology Program Contribu- 
tion No. 264. 

SUMMARY 

1. All individuals of Urosalpin.v cinerea, Urosalpinx cinerea follyensis, and 
Eupleura caudata etterae from which the proboscis was removed, recovered, and 
fully regenerated the proboscis. By the 4th day after proboscisectomy a blastema 
of loose tissue bound the amputated ends of the esophagus, buccal artery, ducts 
of the salivary glands, and the other tissues to the stump of the proboscis. Be- 
tween the 8th and 12th days after the operation, snails had formed minute pro- 
boscides and radulae. Onset of boring of shell by regenerating snails varied from 11 
to 34 days, and took place only after the radula and associated structures were 
developed and functional. The radula is thus an essential component of the mecha- 
nism of shell penetration. Formation of a given length of radula did not trigger 
penetration ; other unexplained factors appear to be responsible. 

2. Although earliest regenerating rachidian and marginal teeth were small and 
misshapen, increase in size and normalization of form was rapid. In time regenera- 
tion of proboscides was complete, and they resembled normal proboscides anatomi- 
cally, histologically, and functionally. Earliest teeth were worn by abrasion, 
indicating that snails began rasping soon after the teeth and odontophore were 
formed. Boreholes excavated by snails with small newly regenerating radulae 
generally corresponded in form and size to those bored by normal snails ; this is 
evidence that the shape and size of the borehole are the products of chemical 
activity of the accessory boring organ rather than the radula. 

3. Histologically the organization of the regenerating odontophoral cartilages 
and associated musculature and other tissues was similar to that seen in regenerating 
vertebrate limbs. In both cases the regenerative process is dependent upon a 
unique cell aggregate (the blastema of vertebrates, and its analogue in muricid 



REGENERATION OF GASTROPOD PROBOSCIS 331 

snails), a cap of cells organized at the regenerating tip of the amputated structure. 
4. The boring habit and rapid regeneration of the proboscis are distinct assets 
in procurement of food, and perhaps account in part for the biological success of 
Urosalpinx cincrea and Euplcnra can data and their significance as major predators 
of commercial oysters. 

LITERATURE CITED 

BUTLER, E. G., 1933. The effects of x-irradiation on the regeneration of the fore-limb of 

Amblystoma larvae. /. E.vp. Zool, 65 : 271-315. 
CARRIKER, M. R., 1943. On the structure and function of the proboscis in the common oyster 

drill, Urosalpin.v cincrea Say. /. Morphol., 73 : 441-506. 
CARRIKER, M. R., 1959. Comparative functional morphology of the drilling mechanism in 

Urosalpinx and Euplcnra (muricid gastropods). Proceedings XV th International 

Congress of Zoology, London, 1959 : 373-376. 

CARRIKER, M. R., 1961. Comparative functional morphology of boring mechanisms in gastro- 
pods. Amcr. Zool., 1 : 263-266. 
CARRIKER, M. R., AND D. VAN ZANDT, 1972. Predatory behavior of a shell-boring muricid 

gastropod. Pages 157-244 in H. E. Winn and B. L. Olla, Eds., Behavior of Marine 

Animals: Current Perspectives in Research, Vol. 1, Invertebrates. Plenum Press, 

New York. 
CARRIKER, M. R., AND E. L. YOCHELSON, 1968. Recent gastropod boreholes and Ordovician 

cylindrical borings. Contrib. Paleontol. Geol. Surv. Prof. Paper. 593-B : B1-B26. 
DEMORAN, W. J., AND G. GUNTHER, 1956. Ability of Thais haeinastoina to regenerate its 

drilling mechanism. Science, 123: 1126. 
HYMAN, L. B., 1967. The Invertebrates, Vol. VI, Molhtsca I. McGraw-Hill Book Co., New 

York, 792 pp. 
ISARANKURA, K., AND N. W. RuNHAM, 1968. Studies on the replacement of the gastropod 

radula. Malacologia, 7: 71-91. 
KIORTSIS, V., AND H. A. L. TRAMPUSCH, 1965. Regeneration in Animals and Related Problems. 

North-Holland Publ. Co., Amsterdam, Netherlands, 568 pp. 
LASH, J. W., AND AL W. WHITEHOUSE, 1960. An unusual polysaccharide in chondroid tissue 

of the snail Busycon: polyglucose sulfate. Biochcm. J., 74: 351-355. 
MATHEWS, M. B., 1968. Molecular evolution of connective tissue. Pages 199-236 in P. 

Person, Ed., Biology of the Mouth. Publication No. 89, American Association for 

the Advancement of Science, Washington, D. C. 
PERSON, P., AND D. E. PHILPOTT, 1963. Invertebrate cartilage. Ann. Neiu York Acad. Sci., 

109: 113-126. 
PERSON, P., AND D. E. PHILPOTT, 19t>9. The nature and significance of invertebrate cartilages. 

Biol. Rev., 44 : 1-17. 
THORNTON, C. S., 1968. Amphibian limb regeneration. Adran. Mnrphol., 7: 205-249. 



Reference: />'/'/. null.. 143: 332-343. ("October, 1 L >72) 



LATITUDINAL EFFECTS ON METABOLIC RATES IN THE CRICKET 

FROG, ACRIS CREPITANS: ACUTELY MEASURED 

RATES IN SUMMER FROGS 

DONALD G. DUNLAP 
Department of Biology, University of South Dakota, Vermillion, South Dakota 

If a poikilothermic animal could not compensate for temperature in metabolic 
activity, the rates of chemical reactions and the rates of various activities of the or- 
ganism could be expected to be proportional to the temperatures to which the 
animal is exposed. It is, however, well documented that a great variety of 
poikilothermic animals can compensate for temperature in various physiological 
activities (Bullock, 1955; Precht, Christophersen and Hensel, 1955; Prosser, 
1964). Northern populations of the same or closely related species often have 
higher rates of activity than southern populations measured at the same temperature. 
As a result, northern and southern populations may have similar rates of activity 
although they are living in very different thermal environments (Bullock, 1955; 
Precht, 1958; Vernberg, 1962). ' 

The compensation for temperature in physiological activities which may be 
encountered among populations from different latitudes is frequently found to be 
stable and the differences are assumed to be genetically determined. On the other 
hand, the response of an individual organism to temperature may be dependent 
upon the temperatures to which the animal was previously exposed. Any such 
acclimation effect must be ruled out before differences between geographically 
separated populations can be considered to have a genetic basis. 

Several investigators have compared the metabolic rates of latitudinally or 
altitudinally separated populations of the same or different species of Anura. 
Thus, Tashian and Ray (1957) reported differences in the rates of oxygen con- 
sumption between adults of northern and southern species of frogs. In addition, 
Tashian and Ray (1957) compared northern and southern subspecies of Bufo 
boreas while Packard (1971) compared montane and piedmont populations of 
Pseudacris triseriata. Significant differences were not found in either case. How- 
ever, Jameson, Taylor and Mountjoy (1970) report a great deal of variability in 
metabolic rates between populations of the frog, Hyla regilla, from localities ex- 
tending from British Columbia to Baja California. They found no clear-cut 
correlation between latitude and metabolic rate but did find that frogs from locali- 
ties which were similar with respect to climate showed similar degrees of metabolic 
adjustment and tended to differ markedly from frogs from climatically different 
regions. 

In the examples cited, the possibilities that genetically fixed latitudinal and 
short-term acclimation effects would be confounded were reduced by comparing ani- 
mals acclimated at the same temperature under controlled conditions in the labora- 
tory. Nonseasonal thermal acclimation of the metabolic rate has been reported 

332 



LATITUDE AND METABOLISM IN ACRIS 333 

for a number of anurans and is often quite pronounced. A partial compensation for 
temperature in the metabolic rate has been reported for several species of Rana 
(Stangenberg, 1955; Rieck, Belli and Blaskovics, 1960; Jankowsky, 1960) and 
for Bufo boreas (Bishop and Gordon, 1957). In these anurans, animals which 
were acclimated at low temperatures were reported to have a higher metabolic rate 
when measured at an intermediate temperature than those acclimated at a higher 
temperature. On the other hand, Rana escnlcnta sampled in the summer (Locker 
and Weish, 1966; Stangenberg, 1955: Table 1) and Acris crepitaus (Dunlap, 
1969, 1971), have been reported to exhibit inverse compensation. In this case, 
frogs acclimated at high temperatures have higher metabolic rates when measured 
at an intermediate temperature than those acclimated at lower temperatures. 

Considering the taxonomic diversity of the Anura and the wide-spread distri- 
bution of the order, there is a paucity of information on the relationship between 
latitude, climate and metabolic rates within the taxon. Furthermore, the majority 
of cases in which latitudinally related effects on metabolic rates have been reported 
have involved animals which exhibit partial compensation (Vernberg, 1962). 
There is, then, a need for more data on which to base hypotheses concerning the 
general significance of inverse compensation to those organisms in which it has 
been reported. Consequently, a comparison was made of the acutely measured 
metabolic rate-temperature curves of acclimated cricket frogs from two latitudinally 
widely separated populations. In this fashion it was expected that any obvious 
differences in acclimation pattern or in the metabolic responses of the two popula- 
tions of frogs to changing temperatures would be demonstrated. 

MATERIALS AND METHODS 

Samples of cricket frogs were collected in July from near Yermillion, South 
Dakota (Latitude 42 48'N, elevation 1,220 ft) and Austin, Texas (Latitude 30 
IS'N, elevation 615 ft). Vermillion lies approximately 870 miles north of Austin. 

Frogs from South Dakota were collected one to two days prior to placing them 
in the acclimation chambers. Texas frogs were collected, shipped to Vermillion via 
air express and placed in the acclimation chambers upon arrival. Groups of frogs 
were acclimated in the dark for 5-7 days (Dunlap, 1969) at experimental tem- 
peratures of 15 and 25 1 C. They were maintained in loosely covered glass 
jars and had access to free water but were not fed during the course of acclima- 
tion. Oxygen consumption was measured for individual frogs using a refrigerated 
Gilson differential microrespirometer equipped with 100 ml flasks. Each flask 
received 5 ml deionized water in the animal chamber and carbon dioxide was 
absorbed by 1.5 ml 20% KOH placed in the side arm. The flasks were equilibrated 
for 30 min and readings were taken every 15 min for two hours. Stability of the 
system was routinely monitored by the insertion of a blank specimen vessel. 

Metabolic rates are given as /A/g per hr STP of oxygen and are based on the 
average hourly uptake over the two hour period. These can best be considered as 
routine rates in the sense of Fry (1957) inasmuch as there was no control of 
spontaneous locomotor activity during the two hour period of determination. 
Measurements of oxygen consumption which are made within a few hours of 
transfer of the frogs from the acclimation temperature to the temperature of determi- 
nation referred to as acute measurements and the- corresponding rates as acute 



334 



DONALD G. DUNLAP 



rates following the terminology of Bullock (1955). Acclimated rates refer to 
rates calculated for animals in which the acclimation temperature and the tem- 
perature of determination are the same. 

In the determination of the acutely measured metabolic rate-temperature (R-T) 
curves, oxygen consumption was measured at one of seven temperatures (5, 10, 15. 
20, 25, 30 or 35 ; b 0.1 C) for frogs acclimated at 15 or 25 1 C. Each combi- 
nation of acclimation temperature and determination temperature for each of the 
two localities was represented by a sample of six frogs. A different group of frogs 
was used for each set of determinations and each frog was used only once. These 
data, then, are based on 84 different frogs from each of the two localities or a total 
of 168 frogs. 

Animals collected from each locality were assigned to each of the individual 
groups on a random basis with the restrictions that there should be approximately 
equal proportions of males and females and equal proportions of three arbitrarily 
assigned size classes within each group. Frogs from South Dakota averaged 
larger (1.5 g body wt) than those from Texas (0.5 g). The statistical techniques 
used in the analyses are from Li (1957) and Ostle (1963). 



TABLE I 

Mean metabolic rates in pl/g per hr and their standard errors for Acris crepitans/row South Dakota 

and Texas. The frogs were acclimated at 15 and 25 C and determined at the temperatures 

indicated. Each mean is based on a sample of six frogs. The data have not been 

corrected for differences in body weight 



Locality 


Acclimation 

temp. 


Determination temperature 


S 


10 


15 


20 


25 


30 


35 


South 
Dakota 


15 


35 1.0 


67 8.1 


77 5.9 


108 7.8 


160 8.4 


236 12.4 


349 15.9 


25 


56 3.7 


102 7.2 


154 14.8 


169 10.3 


169 9.6 


249 16.8 


332 11.9 


Texas 


15 


33 2.1 


66 5.6 


67 6.6 


156 13.0 


174 15.2 


226 25.6 


374 37.0 


25 


44 5.0 


80 8.7 


110 6.6 


173 16.5 


234 46.0 


220 15.1 


296 11.8 



RESULTS 

The mean metabolic rates of the Texas and South Dakota frogs are shown in 
Table I. Since the mean body weights of frogs from the two localities were 
markedly different and since metabolic rate may be a weight-dependent variable in 
cricket frogs (Dunlap, 1969, 1971), comparisons among sets of data for any one 
determination temperature were made using a 2 )< 2 analysis of covariance. In each 
analysis, mean metabolic rates are compared for samples of frogs from each locality, 
acclimated at 15 and 25 C and determined at one of the seven temperatures. 
Prior to analysis, the data were subjected to a Iog 10 transformation for both metabolic 
rate and body weight. This transformation has the double effect of reducing the 
heterogeneity of the variances and of transforming the regression lines to a more 
linear form (Dunlap, 1971). Also, prior to the analysis, the hypothesis that the 
regression coefficients of the regression lines being compared are equal was tested 
and accepted (P > 0.1) in all cases). The results of the analyses are shown in 



LATITUDE AND METABOLISM IN ACRIS 



335 



Table II. Interaction was significant (P < 0.05) at determination temperatures of 
20 and 35 C so these sets of data were analyzed further. 

j 

Locality comparisons 

Differences in mean metabolic rates between Texas and South Dakota frogs 
were not significant at a determination temperature of 5 C or 25 C (P > 0.25 in 
each case). At 10 and 15 C, overall significance for locality was borderline 
(P > 0.05. < 0.1). If, however, metabolic rates of frogs acclimated at 25 C are 
compared separately from those of the 15 C frogs, the mean rate of the South 
Dakota frogs is significantly higher (P < 0.05) than that of the Texas animals. 
There is no significant difference attributable to locality when both samples are 
acclimated at 15 C and determined at either 10 or 15 C (P > 0.25). At 20 C 



TABLE II 

Mean squires, calculated F-ratios and their associated probabilities resulting from the 2X2 analysis 

of coviriince of mstabolic rates in Acris crepitans following a log transformation of the data. 

The localities compared are Austin, Texas and Vermillion, South Dakota and the 

acclimation temperatures; 15 and 25 C. In each case metabolic rates were 

determined at the temperature indicated. For each analysis, the 

degrees of freedom are 1 and 19. Values in 

parentheses below the F-values 

represent probabilities 



Source of 
variation 


Temperature of determination (C) 


5 


10 


15 


20 


25 


30 


35 


Locality M.S. 
Acclimation M.S. 
Interaction M.S. 
Error M.S. 


0.0021 
0.1890 
0.0005 
0.0111 


0.0387 
0.0993 
0.0204 
0.0091 


0.0313 
0.3462 
0.0135 
0.0100 


0.0069 
0.0809 
0.0348 
0.0074 


0.0005 
0.0148 
0.0054 
0.0145 


0.0466 
0.0010 
0.0008 
0.0048 


0.0253 
0.0242 
0.0128 
0.0025 


F-ratios 
Locality 


0.184 
O0.25) 


4.251 
O0.05; <0.1) 


3.127 
O0.05; <0.10) 


0.908 
O0.25) 


0.037 
( >0.25) 


9.765 
0.01) 


10.138 
( <0.005) 


Acclimation 


16.950 
0-001) 


10.899 
0.005) 


34.629 
(< 0.001) 


10.993 
(< 0.005) 


1.023 
O0.25) 


0.210 
O0.25) 


9.722 
(<0.01) 


Interaction 


0.048 

O0.25) 


2.234 
( >0.10) 


1.350 
O0.25) 


4.733 
( <0.05) 


0.374 
O0.25) 


0.168 
O0.25) 


5.153 
( <0.025) 



only the frogs acclimated at 15 C exhibited a significant difference attributable to 
locality (P < 0.005) with the Texas frogs having the higher rate. For frogs 
acclimated at 25 C and determined at 20 C there is no significant difference at- 
tributable to locality (P > 0.25). At 30 C the metabolic rates of frogs acclimated 
at both temperatures were significantly different for locality, with the South Dakota 
frogs having the higher rates (P < 0.025). At 35 C there is no significant differ- 
ence for the 15 C frogs (P >0.1) but locality differences are significant for the 
25 C frogs (P < 0.025). The South Dakota animals have the higher rate. 

Acclimation comparisons 

The metabolic rates of frogs acclimated at 15 and 25 C are significantly dif- 
ferent at 5. 10 and 15 C (P < 0.005). with the frogs acclimated at 25 C having 



336 



DONALD G. DUNLAP 



500 
400 



'- 300 

_c 



- 200 



c 
o 

1 100 



C 
O 



c 

0) 
O5 
>. 
X 

O 




! 1 



_L 



10 15 20 

Tempera t u re, 



25 



30 



35 



FIGURE 1. Routine oxygen consumption in Acris crcpitans from Texas and South Dakota 
at various temperatures following acclimation at 15 and 25 C. Each point on the graph 
represents the predicted mean metabolic rate for frogs weighing 1 gram. Symbols representing 
the treatments are: Texas, 15 C (); Texas, 25 C (); S. D., 15 C (D) ; S. D., 
25 C (O). 



the higher rates. At 20 C acclimation effects are significant only for the South 
Dakota frogs (P < 0.005), with the 25 C acclimated frogs having the higher 
rates. At 25 and 30 C acclimation effects were not significant (P > 0.25) for 
either locality. At 35 C the South Dakota frogs showed no acclimation effects 
(P > 0.1) but the Texas animals did (P < 0.025). The frogs acclimated at 15 C 
have the higher rate. 

The R-T curves for the four series (Texas frogs acclimated at 15 and 25 C, 
South Dakota frogs acclimated at 15 and 25 C) are shown in Figure 1. The mean 
rates used in the construction of the graph are the metabolic rates for frogs weighing 
1 g predicted from the regression equation calculated from the data of each treat- 
ment. This figure illustrates the strikingly different metabolic responses of frogs 
acclimated at 15 and 25 C when oxygen consumption is measured at temperatures 
of less than 20 C for the Texas frogs and less than 25 C for the South Dakota 
frogs. Warm acclimated frogs from both Texas and South Dakota have a higher 
metabolic rate in this range of temperatures than do cool acclimated animals. The 
magnitude of the acclimation response is, however, greater for the South Dakota 
than for the Texas frogs. This is shown especially well when the R-T curves for 
the frogs acclimated at 25 C are compared. For frogs acclimated at 25 C, South 



LATITUDE AND METABOLISM IN ACRIS 



337 



Dakota frogs have consistently higher metabolic rates than do Texas frogs when 
metabolic rates are determined at 5, 10, and 15 C. At determination temperatures 
above 25 C there are clearcut differences in metabolic responses of frogs from 
the two localities, with the Texas animals having the lower rates. At determination 
temperatures of 30 and 35 C no acclimation effects can be demonstrated for the 
South Dakota frogs. At 35 C, however, the cool acclimated Texas frogs have a 
slightly higher metabolic rate than the warm acclimated frogs. 

Effects of acclimation on sensitivity of metabolic rates to temperature change 

Since the curves of Figure 1 are plotted semilogrithmically, segments of lines 
with equal slopes represent temperature regions with equal O 10 values. O ]0 
values were calculated at 5 C intervals using the equation 



'10 



_/Vi\ 

\vj 



where V l and V 2 are metabolic rates corresponding to the temperatures t x and t... 
The calculated O ]0 values for South Dakota and Texas animals acclimated at 15 and 

TABLE III 

Q\a values determined at 5C intervals for Acris from South Dakota and 
Texas acclimated at 15 and 25C 



Acclimation temperature 


Temperature interval (C) 


5-10 


10-15 


15-20 


20-25 


25-30 


30-35 


South Dakota frogs 
15 

25 


4.109 
3.478 


0.729 
2.226 


2.512 
1.171 


3.066 
0.956 


1.621 
2.582 


3.857 
1.932 


Texas frogs 
15 

25 


2.759 
2.019 


1.395 
3.349 


5.774 
2.256 


1.107 

1.010 


1.241 
1.006 


2.782 
2.226 



25 C are shown in Table III. Chemical reaction rates are usually more than 
doubled per 10 C increase in temperature (Prosser and Brown, 1961). Hence, 
a Q 10 of less than 2 would indicate a relative insensitivity of metabolic rate to tem- 
perature change within the temperature range indicated. As might be surmised 
from an examination of the R-T curves, Q Ift values of approximately 1.0 obtain 
between 15 and 25 C for the South Dakota frogs acclimated at 25 C and between 
20 and 30 C for the 25 C Texas frogs. When frogs from both localities are accli- 
mated at 15 C, Q 10 values are low between 10 and 15 C. In the Texas animals 
the Q u , is low between 20 and 30 C as well. The latter plateau on the 15 C R-T 
curve is barely noticeable for the data from South Dakota frogs. For frogs accli- 
mated at 15 C, segments of the R-T curves characterized by very low O 10 values 
are often immediately preceded by segments with high values, e.g., the curve for 
Texas frogs between 20-30 C. In the R-T curves for frogs acclimated at 25 C, 
on the other hand, the transition between segments with high and those with low 



X DONALD G. DUNLAP 

O 10 values is less abrupt. The acclimated R-T curves for South Dakota and 
Texas Acris between 15 and 25 C are essentially identical and have a Q 10 
value of 2.8. 

DISCUSSION AND CONCLUSIONS 

Although the forms of the acutely measured I\-T curves are complex, the curves 
of the South Dakota and Texas frogs in July show many similarities. They are also 
similar in form to those of South Dakota frogs determined in late May and early 
June (Dunlap, 1971). In each case, the same basic acclimation pattern is found. 
In Precht's (195S) terminology, the metabolic rates of Acris exhibit type V (in- 
verse) compensation when the rates of warm acclimated (25 C) frogs are compared 
to those of cold acclimated (15 C ) frogs at the lower temperature. Type IV (no) 
compensation, however, is evident when the metabolic rates of cold acclimated frogs 
are compared to those of warm acclimated animals at the higher temperature. This 
pattern is reflected in the intersection of the R-T curves which occurs at 20 C for 
the Texas frogs and 25 C for the South Dakota frogs. At temperatures below the 
intersection, warm acclimated frogs have a higher rate of oxygen uptake than do 
the cold acclimated animals. For South Dakota frogs, no acclimation effects are 
evident at the intersection of the R-T curves or at higher temperatures. Acclima- 
tion effects are absent between 20 and 30 C for Texas frogs. At 35 C, however, 
the cold acclimated Texas frogs have slightly higher rates than the warm acclimated 
frogs. As a result of this complex pattern of acclimation, the metabolic rates of 
warm acclimated frogs remain stable and Qi () values approximate 1 over a range in 
body temperature of at least 10 C. See Dunlap (1971 ) for a more detailed anal- 
ysis of this acclimation pattern. 

Cricket frogs are active both night and day and during the day may frequently 
be encountered basking in the sun at margins of ponds and small streams. Fitch 
(1956) reported that in northeastern Kansas, of 102 body temperatures recorded 
for Acris throughout the year, over half were between 28.0 and 31.7 C. In cool 
weather he found that body temperatures often exceeded air temperature by 10 C 
or more, but in warm weather the frogs basked less and body temperatures were 
usually nearer air temperature. Brattstrom (1963), on the basis of his own and 
Fitch's data, suggested that Acris crcpitans may engage in thermoregulatory move- 
ments involving alternate basking and immersion in the water during the day and 
repeated immersion at intervals during the evening while water temperatures are 
higher than ambient air temperatures. The body temperatures of the frogs fluctuate 
less both daily and seasonally than might be expected on the basis of ambient tem- 
perature alone. Thus, Brattstrom (1963) measured body temperatures of Texas 
Acris between 0900 and 2300 hr and reported a mean body temperature of 24.9 C 
with a range from 30.0-22.0 C. During the same period, air temperature varied 
from a high of 27.0 C in the afternoon to a low of 21.5 C at night and water 
temperature varied from 27.2 C in the afternoon to 23.8 C at night. These data, 
then, suggest that Acris can, by behavioral thermoregulation, maintain a body tem- 
perature close to 30 C while the sun is shining. At night, however, body tempera- 
tures drop to levels that, on the average, lie between ambient air and water 
temperatures. 



LATITUDE AND METABOLISM IX .1C HIS 339 

As we have seen, Texas frogs acclimated at 25 C are metabolically relatively 
insensitive to temperature change between 20 and 30 C. All of the values for body 
temperature given by Brattstrom and the bulk of Fitch's warm season values fall 
within these limits. 

In an earlier paper (Dunlap, 1971), I suggested that inverse compensation to- 
gether with the associated regions of metabolic insensitivity to temperature change 
enabled warm acclimated summer animals to maintain a high and relatively stable 
metabolic rate in spite of fluctuations in body temperature which occur between day 
and night and from day to day. Conversely, under conditions of constant and low- 
temperature, as could be expected to occur during hibernation, the frogs would be 
cold acclimated and the relatively low rate of metabolism would result in a reduced 
rate of energy expenditure from energy reserves during the dormant period. The 
above interpretation is consistent with the available data on the body temperature 
of cricket frogs under field conditions. This model is based on the assumption that 
the frogs are warm acclimated throughout the summer. That is, they acclimate to 
their basking temperature or to a mean body temperature rather than to the lower 
temperature reached during the daily thermoperiod. Although I am aware of no 
experimental evidence for this with respect to metabolic acclimation, the work of 
Hutchison and Ferrance (1970) and Seibel (1970) lends credence to such an as- 
sumption. These investigators reported that in the frog, Rana pipicns. the critical 
thermal maximum responds to the maximum temperature when the frog is sub- 
jected to a daily thermoperiod. 

In comparing the R-T curves of the 25 C acclimated frogs from South Dakota 
and Texas, it may be seen that the region of relative metabolic insensitivity to tem- 
perature extends from 20 to 30 C for the Texas animals and from 15 to 25 C for 
the South Dakota animals (Fig. 1). Furthermore, the metabolic rates of warm 
acclimated South Dakota frogs are higher than those of Texas frogs at all tested 
temperatures below 20 and above 25 C. These differences would seem to be ac- 
counted for by a simple translation of the R-T curve of the South Dakota frogs 5 
to the left as compared to the Texas population. If the warm acclimated curve for 
the South Dakota frogs is moved 5 C to the right the two curves are essentially 
superimposed. 

The mean maximum and minimum temperatures for July are 31.5 and 17.2 C, 
respectively, for Vermillion, South Dakota (Spuhler, Lytle and Moe, 1967) and 
34.6 and 23.2 C for Austin, Texas (Blood, 1960). The differences noted above 
between the warm acclimated R-T curves of the northern (South Dakota) and the 
southern (Texas) populations, taken with the climatological data are consistent with 
the hypothesis of the role of metabolic patterns in the maintenance of metabolic 
stability in a varying thermal environment. If the thermoregulatory abilities of the 
two populations of frogs are similar, one could expect that in July the nocturnal body 
temperatures of the South Dakota frogs would, on the average, be lower than those 
of the Texas frogs. Yet, due to the shift of the curves for the South Dakota frogs 
to the left, nocturnal metabolic rates could be expected to average about the same 
for the two populations. Conversely, the mean maximum body temperatures of the 
Texas frogs might be expected to average somewhat higher during the day than 
those of the South Dakota animals. Under these conditions the depression of the 
metabolic rates of the' Texas frogs at higher temperatures relative to the South 



340 DONALD G. DUNLAP 

Dakota frogs would contribute toward the stabilization of the metabolic rates at 
higher body temperatures. 

Except for the 20 C determination temperature, South Dakota and Texas frogs 
acclimated at 15 C do not differ significantly from each other. At 20 C the 15 C 
Texas animals behave metabolically as warm acclimated animals while the 15 C 
South Dakota frogs still have depressed rates. This difference in metabolic rates 
between samples from the two localities, if it proves to be characteristic of winter 
as well as summer animals, might be related to differences in degrees of winter 
activity of frogs at the two localities. In the Vermillion area Acris spends four to 
five months hibernating in ponds with body temperatures probably approximating 
3 C (Dunlap, 1971). In the vicinity of Austin, frogs are active during all months 
of the year although the number of active frogs decreases in December and January 
(Pyburn, 1958). At temperatures above 5 C Dunlap (1971) found no significant 
differences in the metabolic rates of frogs acclimated at 5 and 15 C. If the acclima- 
tion pattern remains constant throughout the year, the abrupt increase in metabolic 
rate between 15 and 20 C in the Texas frogs would allow cold acclimated winter 
frogs to attain high metabolic rates and presumably high activity rates on warm 
sunny winter days. On the other hand, the South Dakota frogs would retain the 
depressed metabolic rates even in the face of brief periods of warm weather. If the 
northern populations were active during the occasional warm winter periods when 
the ice melts they would still be faced with a scarcity of food and the danger of being 
excluded from the hibernation pools when freezeup occurs again. Consequently, 
the greater constraints placed on the attainment of a high metabolic rate in the 
northern as compared to the southern population at moderate temperatures is, it 
seems to me, consistent with an adaptive interpretation of the metabolic and acclima- 
tion pattern. 

The acclimated R-T curves between 15 and 25 C are almost identical for the 
two populations and with a Qio of 2.8 exhibit no metabolic compensation for tem- 
perature. Dunlap (1971) has suggested that this may relate to the fact that these 
small frogs live in an environment of pronounced thermal instability. Consequently, 
body temperature could be expected to equal acclimation temperature for only a part 
of the time that the frog is active. This is, of course, quite different from Bullock's 
(1955) model which applies to environments such as the oceans in which environ- 
mental temperatures are much more stable and change slowly with the seasons. 
Under these conditions Bullock suggested that the acclimated R-T curves would 
exhibit temperature compensation. 

The Texas and South Dakota populations of Acris crcpitans are both placed in 
the subspecies A c. blanchardi ( Conant, 1958). This arrangement is supported 
on the basis of their general morphological (Harper, 1947) and biochemical (Des- 
sauer and Nevo, 1969) similarity. However, populations of frogs from Texas tend 
to be more polymorphic for transferrins and slow esterases than the South Dakota 
populations (Dessauer and Nevo, 1969). Further, the two populations differ with 
respect to the predominance of one or the other of the alleles of the H subunit locus 
of lactate dehydrogenase ( Salthe and Nevo, 1969). The one South Dakota popu- 
lation studied is monomorphic for one form of HLDH while Texas populations tend 
to be either monomorphic for the other form or are polymorphic. This is of special 
interest in view of the growing body of evidence for the function of enzyme variants 



LATITUDE AND METABOLISM IN ACR1S 341 

in minimizing the sensitivity of a given reaction to temperature. Hochachka and 
Somero (1968), for example, suggest that enzyme variants (e.g., LDH enzymes) 
are induced during short-term acclimation and the same variants may be differen- 
tially selected by different populations during evolutionary adaption to different 
regional climates. 

The results of the present study are in general agreement with the studies cited 
above. The South Dakota and Texas frogs show an overall similarity in acclima- 
tion pattern, as reflected in the acutely measured R-T curves and in terms of their 
metabolic rates within the range of temperatures encountered in the course of their 
activities. The differences described here are suggestive of selection acting as fine 
tuning mechanism, adjusting the metabolic rate-temperature relations of the different 
populations to their specific environments. 

I wish to thank Mr. Joe Ideker for supplying the Texas frogs used in this study. 
This investigation was initiated under Grant No. GB-5298 from the National Sci- 
ence Foundation. 

SUMMARY 

Samples of cricket frogs were collected in Texas and in South Dakota in July 
and acclimated for 5-7 days at 15 and 25 C. Routine metabolic rates were deter- 
mined at 5, 10, 15, 20, 25, 30, and 35 C for samples from both localities acclimated 
at both temperatures. Sets of data from samples determined at each of the seven 
determination temperatures were subjected to a 2 X 2 analysis of covariance. 

For both localities, warm acclimated (25 C) frogs had higher metabolic rates 
than cool acclimated (15 C) frogs at determination temperatures below 20 C 
(Texas) or below 25 C (South Daokta). At determination temperatures from 
25 to 35 C (South Dakota) and 20 to 30 C (Texas), acclimation effects were not 
significant. At 35 C, Texas frogs acclimated at 15 C had a higher metabolic rate 
than those acclimated at 25 C. 

Frogs acclimated at 15 C showed no significant locality effects when determined 
at 5, 10, 15, 25, and 35 C. At 20 C, the Texas frogs acclimated at 15 C had a 
significantly higher rate than the corresponding South Dakota frogs, while at 30 C, 
the South Dakota frogs had the higher rate. When both were acclimated at 25 C, 
the South Dakota frogs had a significantly higher rate than those from Texas at 
determination temperatures of 10, 15, 30, and 35 C. At 5, 20, and 25 C, how- 
ever, no significant locality effects were apparent. 

On the acutely measured R-T curves of cricket frogs acclimated at 25 C, there 
is a region of pronounced metabolic insensitivity to temperature in which region 
the Qio approximates 1.0. The region lies between determination temperatures of 
15-25 C for the South Dakota frogs and between 20-30 C for the Texas animals. 
Frogs from both localities acclimated at 15 C have a low Qio value between 
10-15 C and the Texas frogs have another one between 20 and 30 C. The dif- 
ferences noted above between the w^arm acclimated R-T curves of the northern and 
southern populations, taken in conjunction with published data on body temperature- 
environmental temperature relationships in Acris and with published climatological 
data, are consistent with the hypothesis of the role of metabolic patterns in the 
maintenance of metabolic stability in a varying thermal environment. 



342 DONALD G. DUNLAP 

LITERATURE CITED 

BISHOP, L. G., AND M. S. GORDON, 1967. Thermal adaptation of metabolism in anuran amphib- 
ians. Pages 263-280 in C. L. Prosser, Ed., Molecular Mechanisms of Temperature 

Adaptation. American Association for the Advancement of Science, Washington. 
BLOOD, R. D. W., 1960. Climates of the States; Texas. U. S. Dept. Commerce Weather Bu- 
reau, Climatography of the United States No. 60-41. U. S. Government Printing Office, 

Washington, 28 pp. 
BRATTSTROM, B. H., 1963. A preliminary review of the thermal requirements of amphibians. 

Ecology. 44: 238-255. 
BULLOCK, T. H., 1955. Compensation for temperature in the metabolism and activity of poikilo- 

therms. B'wl. Rn:. 30 : 311-342. 
CONANT, R., 1958. A Field Guide to Reptiles and Amphibians. Houghton MifHin, Boston, 

366 pp. 
DESSAUER, H. C., AND E. NEVO, 1969. Geographic variation of blood and liver proteins in cricket 

frogs. Biochcm. Genet., 3: 171-188. 
DUNLAP, D. G., 1969. Influence of temperature and duration of acclimation, time of day, sex 

and body weight on metabolic rates in the hylid frog, Acris crepitans. Comp. Biochem. 

Physiol., 31 : 555-570. 
DUNLAP, D. G., 1971. Acutely measured metabolic rate-temperature curves in the cricket frog, 

Acris crepitans. Comp. Biochcm. Physiol.. 38A : 1-16. 

I-'IHH, H. S., 1956. Temperature responses in free-living amphibians and reptiles of north- 
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FRY, F. E. J., 1957. The aquatic respiration of fish. Pages 1-63 in M. E. Brown, Ed., The 

Physiology of Fishes, Vol. 1. Academic Press, New York. 
HARPER, F., 1947. A new cricket frog (Acris) from the middle western states. Proc. Biol. Soc. 

Washington, 60 : 39-40. 
HOCHACHKA, P. W., AND G. N. SOMERO, 1968. The adaptation of enzymes to temperature. 

Comp. Biochem. Physiol.. 27 : 659-668. 
HUTCHISON, V. H., AND M. R. FERRANCE, 1970. Thermal tolerances of Rana pipiens acclimated 

to daily temperature cycles. Herpctologica. 26: 1-8. 
JAMESON, D. L., W. TAYLOR AND J. MOUNTJOY, 1970. Metabolic and morphological adaptation 

to heterogenous environments by the Pacific tree toad, H\la reqilla. Evolution, 24: 

75-89. 
JANKOWSKY, H., 1960. L'ber die hormonale Beeinflussung der Temperatur-adaptation beim 

Grasfrosch (Rana tcmporaria L.). Z. Vcral. Physiol., 43: 392-410. 

Lr, J. C. R., 1957. Introduction to Statistical Inference. Edwards Brothers, Ann Arbor, 568 pp. 
LOCKER, A., AND P. WEISH, 1966. Quantitative aspects of cold-adaptation and its thryoxine 

model in cold- and warm-blooded animals. Helgolaender Wiss. Meeresunters., 14: 

503-513. 

OSTLE, B., 1963. Statistics in Research. (2nd Ed.) Iowa State University Press, Ames, 585 pp. 
PACKARD, G. C., 1971. Oxygen consumption of montane and piedmont chorus frogs (Pseudacris 

triscriata) : a study of evolutionary temperature compensation. Physiol. Zool., 44: 

90-97. 
PRECHT, H., 1958. Concepts of the temperature adaptation of unchanging reaction systems of 

cold-blooded animals. Pages 50-77 in C. L. Prosser, Ed., Physiological Adaptations. 

American Physiological Society, Washington. 
PRECHT, H., J. CHRISTOPHERSEN AND H. HENSEL, 1955. Temperatur and Leben. Springer, 

Berlin, 514 pp. 
PROSSER, C. L., 1964. Perspectives of adaptation: theoretical aspects. Pages 11-25 in D. B. 

Dill, Ed., Handbook of Physiology, Adaptation to the Environment. American 

Physiological Society, Washington. 
PROSSER, C. L., AND F. A. BROWN, 1961. Comparative Animal Physiology. (2nd Ed.) W. B. 

Saunders, Philadelphia, 688 pp. 
PYBURN, W. F., 1958. Size and movements of a local population of cricket frogs (Acris 

crepitans). Texas J. Sci., 10: 325-342. 
K'IKCK, A. F., J. A. BELLI AND M. E. BLASKOVICS, 1960. Oxygen consumption of whole animals 

and tissues in temperature acclimated amphibians. Proc. Soc. Exp. Biol. Med., 103 : 

436-439. 



LATITUDE AND METABOLISM IN ACR1S 343 

SALTHE, S. N., AND E. NEVO, 1969. Geographic variation of lactate dehydrogenase in the cricket 

frog, Acris crcpitans. Biochcm. Genet., 3: 335-341. 
SEIBEL, R. V., 19.' 0. Variables affecting the critical thermal maximum of the leopard frog, 

Rana pipicns Schreber. Herpetologica, 26: 208-213. 
SPUHLER, W., W. F. LYTLE AND D. MOE, 1967. Climatoloyical Summary No. 8; Ver million, 

South Dakota. Agricultural Experiment Station, South Dakota State University, 

Brookings, 6 pp. 
STANGENBERG, G., 1955. Der Temperatureinfluss auf Lebensprozesse und den Cytochrom 

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320-332. 
TASHIAN, R. E., AND C. RAY, 1957. The relation of oxygen consumption to temperature in 

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of animal populations. Ainni. Rcr. Physiol.. 24: 517-546. 



Reference: Biol. Bull., 143: 344-357. (October, 1972) 



HYPERBARIC OXYGEN AND EMBRYONIC DEVELOPMENT IN 

ARBACIA PUNCTULATA x 

PAUL M. HEIDGER, JR., ROBERT G. SUMMERS, 2 AND JAMES A. MILLER, JR. 

Department of Anatomy, Tulane University School of Medicine, Neiv Orleans, Louisiana 70112, 
and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543 

Oxygen toxicity to living systems has been recognized since the classical studies 
of Bert (1878), but relatively little attention has been directed toward determining 
the effects of oxygen at high pressure upon developing systems. Such studies do 
include those of New and Coppola, 1970 (rat) ; Pizzarello and Shircliffe, 1967 
(chick) ; Perm, 1964 (hamster) ; Rosenbaum, 1960, Malamed, 1957, Nelson, 1949, 
and Rauber, 1884 (frog); and Rosenbaum and Wittner, 1960 (sand dollar). 
Miller, Miller, DeSha and Heidger (1969) investigated the effects of hyperbaric 
oxygen (HBO) upon embryonic development in the hydroid, Tubularia, and 
demonstrated that differentiation is blocked by exposure to pure oxygen at pres- 
sures of 2 to 4 atmospheres absolute (AA). Blockage of differentiation was ac- 
companied by inhibition of succinic dehydrogenase activity. In view of the high 
succinic dehydrogenase activity which prevails during portions of sea urchin de- 
velopment (Gustafson and Hasselberg, 1951), it was of interest to extend our 
investigation to another phylum, the Echinodermata, and to a species from which 
large numbers of embryos are readily obtained and are routinely reared in vitro. 
This paper presents the results of studies of the effects of hyperbaric oxygen upon 
the embryonic development of Arbacia from fertilization to the time of formation 
of the pluteus larva. 

MATERIALS AND METHODS 

Specimens of Arbacia punctnlata collected by the Supply Department of the 
Marine Biological Laboratory were used in all experiments. Gametes were shed 
by electrical stimulation (Harvey, 1956), and the eggs, pooled from several fe- 
males, were suspended in filtered sea water at room temperature and were washed 
several times. They were inseminated with a dilute sperm suspension. The 
percentage fertilization was assessed 10 minutes post-insemination by observation of 
the elevation of the fertilization membrane, and all samples showing less than 95% 
fertilization were discarded. 

The fertilized eggs were then distributed to finger bowls or Petri dishes such 
that a single layer of zygotes was distributed over the bottom of the container. The 
zygotes then were transferred to a 16 C constant temperature room, and were 
assigned randomly to control or experimental groups. Control groups were al- 
lowed to develop in air at 16 C, and experimental groups were subjected at the 

1 Supported by NIH Grant 5T1 GM 00793 to J. A. Miller and a Faculty Research Award 
from the University of Maine to R. G. Summers. 

2 Present address : Department of Zoology, University of Maine, Orono, Maine 04473. 

344 



HYPERBARIC OXYGEN AND DEVELOPMENT 345 

same temperature to 100% oxygen at 3 AA, within a Bethlehem hyperbaric 
chamber. Compression and decompression of the chamber were performed 
slowly such that no temperature changes occurred. The chamber was thoroughly 
flushed with gas before each compression. All times reported in this study were 
recorded from the time of insemination, taken to correspond closely with the time 
of fertilization. 

Sampling 

Sampling was accomplished by two means. In the case of a continuing culture 
of embryos, an aliquot of embryos and sea water was withdrawn by pipette after 
the entire culture had been agitated by a stream of air. In the case of terminal 
samples, the entire culture was fixed by the addition of several ml of 5% 
glutaraldehyde in sea water after a sample had been removed for assessment of 
viability. Two investigators routinely checked the samples by the double blind 
technique. Staging of embryos was carried out according to Harvey, 1956. 

Histological studies 

Control and experimental embryos were fixed by immersion in 3% glutaralde- 
hyde in sea water for 24 hours. The embryos were washed in filtered sea water, 
dehydrated in alcohols, cleared in xylene, and embedded in Tissuemat (Fisher). 
Seven-micron sections were cut and were stained with Ehrlich hematoxylin and 
alcoholic eosin-Y. Both embryos and histologic preparations were examined and 
photographed using a Reichert Zetopan photomicroscope. 

Three principal series of experiments were performed: Series 1, To determine 
the effect upon developmnt of constant exposure to hyperbaric oxygen. Embryos 
were exposed to 3 atmospheres absolute oxygen for 72 hours, post-fertilization age. 
Controls developed in air at normobaric pressure. Series 2, To determine the re- 
versibility of effects found in Series 1 experiments. Embryos were exposed to 3 
atmospheres absolute oxygen for 12, 24, 36 or 48 hours. They were removed 
from the hyperbaric chamber and were then allowed to develop in air for up to 
144 hours post-fertilization age. Controls developed in air at normobaric pres- 
sure. Series 3, To determine the time during development at which hyperbaric 
oxygen first exerts its effect. Embryos were allowed to develop in air for 12, 18, 
24, or 36 hours and were then subjected to hyperbaric oxygen until 72 hours, post- 
fertilization age. Controls developed in air at normobaric pressure. 

Special controls 

In addition to the controls outlined above, two series of controls were performed 
for the following reasons. 

Control series 1. To exclude the possibility that observed results might be 
caused by exposure to 100% oxygen (Linde), and not by oxygen under pressure, 
embryos were incubated under 1 A A pure oxygen for 72 hours. 

Control series 2. To exclude the possibility that observed results might be 
caused by increased pressure, and not to oxygen under pressure, embryos were 
incubated in the hyperbaric chamber under 1 AA pure oxygen, and 2 AA pure 
nitrogen for 72 hours. 



346 



HEIDGER, SUMMERS, AND MILLER 



RESULTS 

The key developmental stages and the time at which each was observed during 
development at 16 C are as follows: early blastula, 6 hours; hatching blastula, 12 
hours; mesenchyme blastula. 24 hours; gastrula, 30 hours; late gastrula-early 
prism, 36 hours; and young plutei, 48 hours (see Figs. 9, 10 and 11). 

Series 1 e.vperiinaifs 

The results of this series are summarized in Table I. Both control and 
hyperbaric groups developed in synchrony to the gastrula stage of development. 
At the time of late gastrula, however, the hyperbaric embryos appeared arrested 
and did not proceed to form prisms or plutei. Rather, by 48 hours, most of the 
embryos had lost the archenteron structure distinguishing the gastrula, and were, 

TABLE I 

The effect upon Arbacia development of exposure to 3 .1.1 oxygen 
for various lengths of time 







Per cent of sampli-* 


Length of 
exposure 
to 3 AA O 2 


Treatment 


Dead or 
abnormal 


Unhatched 
blastula 


Mesen- 
chyme 
blastula 


Normal 
gastrula 


Abnormal 
Kastrula 
or 
regress in u 


Prism 


Pluteus 














gastrula 






12 hr Control 


6 


94 














Hyperbaric 


5 


95 












24 hr Control 


6 





94 












Hyperbaric 


7 





93 










.16 hr Control 


6 


(l 





94 








Hyperbaric 


6 








80 


14 






48 hr Control 


4 





(I 


2 





7 


87 


Hyperbaric 


5 








1 


94 









* Minimum of 500 embryos counted in each sample. 

as assessed by optical section of whole embryos, "regressing" to a configuration 
reminiscent of the blastula stage, but with a loose, disorganized population of cells 
within the central lumen. This impression which was gained from the examination 
of whole embryos (see Figs. 4 and 5), was verified by histological preparations 
(see Figs. 7 and 8). These figures illustrate that the relationships of the peripheral 
cells of the embryo appear to have been maintained, but that the archenteron struc- 
ture was disorganized and was not identifiable as such. It is noteworthy that in 
each of the samples studied in this series both control and hyperbaric groups con- 
tained comparable numbers of dead or unclassifiable embryos. Therefore, HBO 
did not exert an immediate lethal effect, but produced a specific alteration in 
morphogenetic pattern. However, if the embryos were maintained in HBO con- 
tinuously for 72 hours, the embryos disaggregated and died. 



HYPERBARIC OXYGEN AND DEVELOPMENT 



347 



100 



9C- 



80- 



70- 



50 



30 



20 




II 




I.I 



G R 
36 



G R Pr PI 
48 



G R Pr PI 

60 
HOURS 



R Pr PI 
72 



G R Pr PI 
84 



FII.UKE 1. Bar graphs showing the per cent of embryos at given developmental stages at 
various times following removal of the embryos from 3 AA oxygen at 36 hours post-fertiliza- 
tion age. Abbreviations are: G^gastrula; R regressing gastrula; Pr prism stage; PI 
pluteus. A minimum of 200 embryos was counted at each age. 

Scries 2 experiments 

Embryos were exposed to HBO for 12, 24, 36 or 48 hours and were then re- 
moved to air and subsequent development was observed. 

(a) Embryos removed at 12 or 24 hours. Embryos exposed to HBO during 
the first 12 hours of development only were not altered morphologically, and de- 
veloped at the same rate as did controls. Development was retarded in embryos 
exposed to HBO for the first 24 hours of development only, retardation being evi- 
denced between the time of removal (mesenchyme blastula stage) and pluteus for- 
mation. Xo abnormal morphological alterations were detected, however. By 60 
hours, nearly all such embryos had reached young pluteus stage. This represents 
a delay of 12 hours beyond the time at which all control embryos reached this 
stage (48 hr). 

(b ) Embryos removed at 36 hours. (See Fig. 1) At the time of removal, the 
embryo population consisted almost entirely of morphologically normal gastrulae, 
plus blastula-like forms designated "regressing gastrulae." No prism stages were 
seen; by 48 hours following removal, 17% of the population consisted of prisms, 
and 3% of normal young plutei. The relative numbers of prism and pluteus stages 
which were seen increased through 84 hours, when the experiment was terminated 
with over 90% of the embryos beyond gastrulation stage, i.e.. in prism or pluteus 



348 



HEIDGER, SUMMERS, AND A1ILLER 



stages. The per cent of dead or unclassifiable embryos at all times studied was 
never more than 6% of the total population, and is not separately plotted in the 
text figures. 

(c) Embrvos removed at 48 hours. ( See Fig. 2) At the time of removal from 
HBO, 37% of the embryo population was classified as regressing gastrula. A large 
proportion of the embryos classified as gastrtilae were not normal gastrulae, but 
showed varying degrees of internal disorganization. For purposes of uniformity 
of classification, however, any embryo showing any evidence whatsoever of archen- 
teron structure was placed in the gastrula category. Twenty-four hours following 
removal, a small population (2%') of prism stages was seen. Between 72 and 96 
hours, plutei were formed, and the prism population increased to 11%. The appear- 
ance of the later stages of development was accompanied by a decrease in the re- 
gressing gastrula population from 41 ' '< at 71 hours to 17% at 96 hours. Gastrulae 
at this time appeared normal. The decline in gastrulae and regressing gastrulae 
and the increase in numbers of later embryonic stages continued, until at 144 hours, 
94 % of the population had progressed past the gastrula stage into prisms or plutei. 



100 



90 - 



70 



GO 



50 



30 



20- 



10 




PI 



48 



72 



96 

HOURS 



120 



144 



FIGURE 2. Bar graphs showing the per cent of embryos at given developmental stages at 
various times following removal of the embryos from 3 AA oxygen at 48 hours post-fertiliza- 
tion age. Abbreviations as in Figure 1. A minimum of 200 embryos was counted at each age. 



HYPERBARIC OXYGEN AND DEVELOPMENT 



349 



Series 3 experiments 

Table II summarizes the per cent of embryos reaching developmental stages 72 
hours post-fertilization when exposed to HBO at various times in development. 
Embryos exposed following 12 hours of development in air ( hatching blastula stage) 
gastrulated at the same time as did controls, but formed the blastula-like structures 
termed "regressing gastrulae" and showed early evidence of disaggregating by 72 
hours. Unlike the regressing gastrulae seen in Series 1, rudimentary spicules were 
occasionally observed in this group embedded within the structure of the regressing 
gastrulae. Embryos exposed to HBO at 18 hours (having reached the swimming 
blastula stage), or at 2-1- hours (having reached mesenchvme blastula stage) were 
delayed in development, but showed a low percentage of regressing gastrulae, and 
a high percentage of normal gastrulae, prisms, and plutei at 72 hours. Embryos 
exposed following 36 hours of development in air ( having reached the gastrula 
stage) progressed uninhibited to form young plutei by 48 hours. The plutei from 
this group when observed appeared morphologically normal but swam very slowly 

TABLE II 

/'//c <(>'<'!/ H/)OH Arbacia development of exposure to 3 A A oxygen 
at various post-fertilization r;c\ 



Age when fir-t 



Pcr cent of embryos in stages at 72 hours of age* 



expi !-<! to 3 \.\ 












oxygen 


Dead 


gasl Mila 


Ki-mrssing 


Prism 


Pluteus 


12 hours 


5 


5 


90 








18 hours 


1 


24 


9 


65 


1 


24 hour- 


2 


22 


7 


59 


10 


36 hour- 


3 








2 


95 



* A minimum of 200 embryos was counted for each a .ye. 



in comparison with controls. Further, normal, young plutei treated with HBO at 
48 hours post-fertilization age were observed at 72 hours to swim much more slowly 
than did controls. These latter observations may possibly reflect a general depres- 
sion of metabolic activity caused by HBO. 

Controls 

Embryu.s reared in 1 AA pure oxygen or in 1 AA pure oxygen plus 2 AA nitro- 
gen did not differ in morphology or in developmental rate from embryos reared in 
air at normobaric pressure (see Figs. 11 and 12). 

DISCUSSION 

The patterns of effects on development of HBO are unique in the two species 
which we have studied. In contrast with toxic agents which produce death, dif- 
ferential inhibition, differential tolerance, conditioning or recovery depending upon 
the concentration and duration of the exposure (Child. 1Q41 ), hyperbaroxia appears 



350 



HEIDGER. SUMMERS, AXU .MILLER 










FIGURES 3-5. 



HYPERBARIC OXYGEN AND DEVELOPMENT 351 

to block development totally for extended periods of time and yet permits total or 
nearly total reversibility in its effects. The reversibility in Tubularia development 
is striking with total blockage of differentiation for a period of as much as five days 
followed by the formation of completely normal actinula larvae (Miller et al. 1969). 
In Arbacia, exposures which arrested gastrulation and induced apparent dedifferen- 
tiation and regression of the developing mesoderm and entoderm never-the-less were 
followed by complete recovery of normal morphology in more than 90% of the 
embryos. There were virtually no inhibited larvae with reduced oral lobes such as 
are characteristic of embryos exposed to KCN, CuSO 4 , LiCl and many other toxic 
agents (cj. Fig. 47, page 199, Child, 1941;. 

This suggests that the blockage which \vas produced by HBO did not induce 
the production of toxic substances which altered any future differentiation. Instead, 
it appears that, although blocked temporarily, their biochemical systems were not 
permanently injured, and could redifferentiate as soon as the excess oxygen was 
removed. These findings are consistent with the concept that the fundamental 
effect of hyperbaric oxygen is to oxidize enzymes which are active in the reduced 
state but are inactivated when in the oxidized condition (Haugaard, 1968). 

Hyperbaric oxygen has been shown to inactivate sulfyhydryl-containing en- 
zymes including succinic dehydrogenase (Haugaard, 1968; Davies and Davies, 
1965). It is significant that this enzymatic activity has been shown to he high 
during gastrulation in the sea urchin (Gustafson and Hasselberg, 1951). A 
second enzymatic activity which is high in activity during gastrulation in the sea 
urchin is that of cathepsin II, which is low in activity up to the mesenchyme 
blastula stage, but which undergoes a sharp rise after this stage (Gustafson and 
Hasselberg, 1951). This enzyme is considered sulfhydryl-dependent (Fruton, 
Irving and Bergmann, 1941) and is known to be inhibited by HBO (Davies and 
Davies, 1965; Rosenbaum, 1960). Furthermore, it has been demonstrated that 
the flavoprotein, diaphorase, is inactivated by oxidation (Williams, 1965). 
Diaphorase-dependent dehydrogenase activities, such as those of glucose-6-phos- 
phate dehydrogenase and malic dehydrogenase, might thus be indirectly inhibited 
under HBO. In this regard, it is significant that glucose-6-phosphate dehydro- 
genase has been shown in Paracentrotus to be maximally active immediately 
preceding, and through the first half of gastrulation (Backstrom, 1959) ; malic 
dehydrogenase activity rises during the mesenchyme blastula stage, gastrulation 
and later stages, paralleling the rise in succinic dehydrogenase activity (Gustafson 
and Hasselburg, 1951). The blockage of such enzymatic activities during develop- 
ment would be expected to disrupt development during the stages at which they 
are highly active; this correlates well with our observations of blockage within 
gastrula stage of HBO-treated embryos. Direct biochemical analysis of enzymatic 
activity during the course of HBO treatment of sea urchin embryos is in progress. 

FIGURE 3. Control gastrulae, 36 hours post-fertilization age. Embryos were reared in air 
at normobaric pressure at 16 C ; whole mount. Scale on all photomicrographs equals 50 fj.. 

FIGURE 4. Embryos reared in HBO at 3 AA, 16 C, for 36 hours : "regressing gastrula" 
stage. Archenteron structure is indistinguishable in these embryos. Compare witli control 
embryos shown in Figure 3 ; whole mount. 

FIGURE 5. Embryo reared in HBO at 3 AA, 16 C, for 48 hours. Morphology is similar 
to the regressing gastrulae shown in Figure 4; no additional morphological changes are ob- 
served with the 12 hours of additional exposure to HBO ; whole mount. 



352 



HEIDGER, SUMMERS, AND MILLER 





' V 




^*-. 



'^5* 
\ + * A. 

* * *> ^ A- 

r. - 



j. ' ' > ^ " 

^ -s ^f ; ,^ "'-'i^ 

'^^^- 'i^t^f 

*V ,* " V v.-%^ 

**-. ^ ^ 




" * 

<- -^-" I 

^^ 








<J8P* 



- 



FIGURES 6-8. 



HYPERBARIC OXYGEN AND DEVELOPMENT 353 

Our studies correlate well with those of Rosenbaum (1960) who reported 
arrest in the gastrula stage in frog embryos treated with HBO, and with those of 
Alalamed (1957) who observed similar blockage in Rana pipicns embryos following- 
incubation of eggs in a medium through which oxygen was bubbled. No studies 
have been made of the protection afforded embryos from the effects of HBO by 
BAL. crysteine, or glutathione (\Yittner and Rosenbaum, 1958a. 1958b). 

Series 1 

Even though the "regressing gastrula" embryos which were observed at both 
36 and 48 hours were indistinguishable morphologically from each other (compare 
Figs. 4 and 5, and Figs, 7 and 8), the 48-hour embryos were much more severely 
inhibited, as evaluated in terms of the time necessary to complete development 
through the pluteus stage (see Figs. 1 and 2). The population of embryos in- 
cubated in HBO for 48 hours required approximately twice the time, following 
removal from the chamber, to complete development through the pluteus stage 
as did those which were exposed to HBO for 36 hours. From observations of 
whole mounts, it appeared that the internal rearrangement of cells during recovery 
of the regressed gastrulae might not be dissimilar to that observed by Giudice and 
Mutolo (1970) in sea urchin embryos reaggregating from dissociated cells. These 
authors have shown that the intestine which develops from reaggregated spheres 
of cells does not form by invagination, as in normal development, but by the 
internal rearrangement of cells about a space destined to become the intestinal 
lumen. However, a detailed histological study of the reversal of inhibition seen 
in the present study remains to be performed. 

Series 2 and 3 

It is instructive to examine the results of Series 2 and 3 in terms of assessing 
the time at which HBO may exert its inhibitory effect. Hyperbaric oxygen 
exerted no evident inhibitory effect upon embryos exposed continuously for 12 
hours following fertilization ; by 24 hours, however, an inhibition to development 
was manifested by the delay in the time of pluteus formation. The inhibition was 
observed to be more severe following 36 or 48 hours and was accompanied by the 
characteristic morphology of the "regressing gastrula." There would appear, 
therefore, to be no residual inhibition of processes regulating pluteus formation 
from only 12 hours of exposure to HBO. Alternatively, those processes essential 
to normal gastrulation and pluteus formation and susceptible to HBO inhibition 
may not be operative at such early stages. These processes may differentiate at 

FIGURE 6. Histological section of control gastrulae reared in air at normobaric pressure 
for 36 hours at 16 C. Note the typically prominent archenteron structure and associated 
mesoderm ; 7-micron section, hematoxylin and eosin stain. 

FIGURE 7. Histological section of "regressing gastrulae." Fertilized eggs were exposed 
continuously to 3 A A oxygen for 36 hours at 16 C. Note the absence of an organized 
archenteron and the "blastula-like" appearance of the embryos ; cf., Figure 6 ; 7-micron section, 
hematoxylin and eosin stain. 

FIGURE 8. Histological section of "regressing gastrulae." Fertilized eggs were exposed 
continuously to 3 AA oxygen for 48 hours at 16 C. Note morphology similar to that in 
Figure 7 above ; 7-micron section, hematoxylin and eosin stain. 



354 



HEIDGER, SUMMERS, AND MILLER 




FIGURES 9-12. 



HYPERBARIC OXYGEN AND DEVELOPMENT 355 

later stages, and be inhibited by exposure to HBO later in development, in the 
mesenchyme blastula stage. It should be noted, however, that HBO may possibly 
exert a generalized depressive effect upon the metabolism of plutei; this is perhaps 
reflected in the slow swimming movements of morphologically normal plutei which 
were evident after prolonged exposure to HBO. 

From Series 3 it may be inferred that embryos which have reached late 
gastrula (36 hr ) prior to exposure to HBO have already been "programmed" 
for pluteus formation, and HBO at 3 AA exerts no effect upon this stage of 
morphogenesis. No information, however, is available in this system concerning 
possible delays in the action of HBO, as is seen in mammals (Haugaard, 1968). 
The possibility of such delays in effective inhibition must be investigated before a 
valid timetable of inhibition may be established. 

Although no detailed comparative study of the initial cleavage patterns of 
control and HBO-treated embryos was made, no obvious effect of HBO upon 
cleavage pattern was noted in the present study, and both HBO-treated and con- 
trol embryos developed synchronously until the time of gastrulation. These findings 
are in accord with those of Rosenbaum and Wittner (1960) who found in 
EdiinaracJiniit* that 4-hour exposures to oxygen at 3 to 4 atmospheres induced no 
anomalies of cleavage or development within the first 8 hours of development. 
Higher pressures of oxygen ( 7 to 8 A A ) did inhibit early cleavage ; later em- 
bryonic development under hyperbaric conditions was not studied. It would be 
of interest to determine whether or not the delay period in inhibition in our 
experiments would be shortened by using oxygen pressures higher than 3 AA. 

In this species, as in our previous experiments with Tubitlaria, (Miller ct a!., 
1969) hyperbaric oxygen has proven to be a reversible inhibitor of development; 
it is hoped that our findings may stimulate the use of HBO in a variety of systems 
as a tool in studies of morphogenesis. 

SUMMARY 

The effects of hyperbaric oxygen (HBO) upon the embryonic development of 
Arbacia were studied from fertilization to the time of formation of the pluteus 
larva. Fertilized eggs were incubated in sea water at 16 C in air or in 3 
atmospheres absolute (AA) pure oxygen in a hyperbaric chamber. Pressure 
control experiments using 1 atmosphere oxygen and 2 atmospheres nitrogen 
demonstrated that the changes observed were caused by elevated pressure of oxygen, 
and not merely to high ambient pressure. Animals exposed continuously to 
hyperbaric oxygen for 48 hours were arrested in the gastrula stage; the archenteron 

FIGURE 9. Normal embryos which have developed in air at 16 C for 36 hours, gastrula 
stage. Compare morphology with that of the inhibited gastrulae shown in Figure 4 ; whole 
mount. 

FIGURE 10. Normal embryos which have developed in air at 16 C for 48 hours, young 
plutei. Compare with embryo which has developed for the same period of time at the same 
temperature, but under 3 AA oxygen, shown in Figure 5 ; whole mount. 

FIGURE 11. Pluteus larvae reared at 16 C under normobaric conditions in air for 72 
hours ; whole mount. 

FIGURE 12. Pluteus larvae reared at 16 C under 3 AA pressure (1 atmosphere oxygen, 
2 atmospheres nitrogen) for 72 hours. Plutei are indistinguishable morphologically from 
embryos reared under normobaric conditions (rf. Fig. 11) ; whole mount. 



356 HEIDGER, SUMMERS, AND MILLER 

was observed to form at 30-32 hours, but to regress, resulting in an unorganized 
mass of cells within the blast ocoel. If removed from HBO at 48 hours, over 90% 
of these inhibited embryis proceeded to form normal prisms and plutei within 144 
hours following removal. In respect to reversibility, therefore, HBO differs 
significantly from many chemical inhibitors of differentiation. 

Embryos reared in HBO following development in air for the first 12 hours 
after fertilization failed to form plutei by 72 hours, whereas controls did so within 
48 hours. Embrxos in which expo>ure to HBO was delayed 18 or 24 hours w r ere 
delayed in reaching the prism or pluteus stage, and showed evidence of dis- 
aggregation within 72 hours post-fertilization. Embryos exposed to HBO follow- 
ing 36 hours of development escaped both the inhibitory and lethal effects of HBO 
and proceeded to form normal plutei by 48 hours. The inactivation by HBO of 
sulfhydryl-containing en/ymes which normally are maximally active during 
gastrulation in the sea urchin mav contribute to the failure of embrvos treated 

o - 1 

with hyperbaric oxygen to complete gastrulation. 

LITERATURE CITED 

BACKSTROM. S., 1959. Activity of glucose-6-phosphate dehydrogenase in sea urchin embryos 

of different developmental trends. /:.r/>. Cell AY.?., 18: 347-35o. 
BERT, P.. 1S7S. La Pressinn Biiroiiietriiiite. Paris. (Barometric Pressure, translated by M. A. 

Hitchcock and F. A. Hitchcock, 1943. College Book Co., Columbus, Ohio). 
CHILD, C. M., 1941. Differential modification of development: Kchinoderms. Pages 197-246 

in Patterns and Problems of Development. University of Chicago Press, Chicago.. 
DAVIES, H. C., AXD R. E. DAVIES, 1965. Biochemical aspects of oxygen poisoning. Pages 

1047-1058 in \V. O. Fenn and H. Rahn, Eds., I latulhouk of l'hysioln,/y. Respiration. 

American Physiological Society, Washington, D.C. 
PERM, Y. H., 1964. Teratogenic effects of hyperbaric oxygen. J'roc. Soc. .r/. Biol. Me<l.. 

116: 975-976. 
FKUTOX, J. S., G. W. IRVIXG ANII M. BERGMAN N, 1941. On the proteolytic enzymes of beef 

spleen, beef kidney, and swine kidney, classification of cathepsins. /. Biol. Chein.. 141 : 

763-774. 
GIUDICE, G., AXD V. MT-TOLO, 1970. Reaggregation of dissociated cells of sea urchin embryos. 

Ad-van. Mvrplwl.,*: 115-158. 
Grs'xAFSOx. T., AXD I. HASSELBERG, 1951. Studies on enzymes in the developing sea urchin 

egg. E.rf. Cell Res., 2 : 642-672. 
HARVEY, E. B., 1956. The American Arhaeia and Other Sea Urchins. Princeton University 

Press, Princeton, New Jersey, 298 pages. 

HAUGAARD, N., 1968. Cellular mechanisms of oxygen toxicity. Physiol. Ret'.. 48: 311-373. 
MALAMED, S., 1957. Gastrular blockage of frogs eggs produced by oxygen at atmospheric 

pressure. Exf. Cell Res., 13 : 391-394. 
MILLER, J. A., JR., F. S. MILLER, D. L. DESHA AXD P. M. HEIDGER, JR.. 1969. Hyperbaric 

oxygen and succinic dehydrogenase in Tubularian development. Biol. Bull.. 137 : 494 

505. 
NELSON, O. E., 1949. The cumulative effect of oxygen-pressure in the blocking of gastrulation 

in the embryo of Rtnni f>if>iens. Anat. Ree., 105 : 599. 
NEW, D. A. T., AXD P. T. COPPOLA, 1 ( )70. Development of explained rat fetuses in hyperbaric 

oxygen. Teratology. 3 : 153-161. 
PIZZARELLO, D. I., AXD A. C. SniRCLiFFE, 1967. Hyperbaric oxygen toxic effects in chick 

embryos. Amcr. Snrg., 33 : 958-959. 
RAUBER, A., 1884. Uber den Einfluss der Temperatur, des atmospharischen Druckes und 

verschiedener Stoffe und die Entwicklung theirischer Eier. Sitsungsber. Naturforsch. 

Gescllschaft. Leipzig, 10 : 55. 



HYPERBARIC OXYGEN AND DEVELOPMENT 



35' 



ROSEXBAUM, R. M., 1960. Gastrular arrest and the control of autulytic activity in the egg 
of Rana pipiens: the comparative effects of oxygen, supramaximal temperature, and 
dinitrophenol. Develop. Bio!., 2 : 427-445. 

ROSENBAUM, R. M., AND M. WiTTNER, 1960. The effects of hyperatmospheric oxygen con- 
centrations on early cleavage in the sand dollar, Echinaracliniits panna. E.rp. Cell Res., 
20 : 416-427. 

WILLIAMS, C. H., JR., 1965. Studies on lipoyl dehydrogenase from Escherichia coli. /. Biol. 
Chem., 240 : 4793-4800. 

WITTXER, M., AND R. M. ROSEXBAUM, 1958a. Reversibility of the inliibitory effects of high 
oxygen concentrations in the development of Raua pipietis. Anat. Ree., 131: 611. 

WITTNER, M., AXD R. M. ROSEXBAUM, 1958b. Resistance and susceptibility to high oxygen 
pressures in the early development of the frog, Raim pipiens. Phvsiol. Zool., 31 : 
294-303. 




Reference : Biol. Bull.. 143: 358-366. (October, 1972) 



SOME FACTORS CONTROLLING REPRODUCTION IN THE 
SPIDER CRAB. LIBINIA EMARGINATA 1 

GERTRUDE W. HINSCH = 

Institute for Molecular and Cellular Evolution and Department of Blolo</y. University of Miami. 

('oral Gahles, Florida 33134 and Marine Biological Laborator\. 

ll'onds Hole. Massachusetts 02543 

While oogenesis in crustaceans has frequently heen studied, detailed knowledge 
of the anatomy, histology and hormonal controls involved in reproduction in female 
brachyurans have been limited (see Ryan, 1967; Adiyodi and Adiyodi, 1970 for 
review). The structure and function of the reproductive system in the crab 
Portitnus during the molting and reproductive cycles of the preadult and two adult 
in stars has been examined recently (Ryan, 1967). Although a definite relation- 
ship exists between the molting and reproductive cycles in crustacean females, little 
is known regarding this relationship between molting and sexual maturation among 
the members of the family Majidae to which Lihinia belongs. For instance, Maja 
is reported to undergo a final or terminal molt ( Drach, 1939). Whether this occurs 
in all Oxyrhyncha is not known. 

This study was undertaken to investigate the relationship between molting and 
sexual maturation and to determine what if any hormonal controls may be operating 
in ovarian development in the female spider crab, Lihiniit ciiiari/hiafa. 

MATERIALS AND METHODS 

Immature and mature- female specimens of Libinia were collected by the Supply 
Department of the Marine Biological Laboratory, Woods Hole, Massachusetts dur- 
ing the summers of 1967-1970. The crabs were maintained in aquaria with running 
sea water and fed periodically on Mvtilus or Spisnla. Carapace length of all crabs 
studied was measured with calipers. 

The eyestalks of immature and mature female crabs were removed at their base 
with a dissecting needle and scissors to induce molting. No special measures were 
needed to control or prevent excessive bleeding. In addition, mature females with 
or without broods were subjected to eyestalk ablation and the effects on reproduction 
were sttulied. Control animals ( non de-eyestalked ) were kept in adjacent aquaria. 
Males were placed in all aquaria. 

Reproductive tracts of both immature and mature females were dissected for 
study. The ovaries were fixed in calciiim-formol, dehvdrated and embedded in 
paraffin for light micro.scupy or were fixed in Karnovsky's ( 1%5 ) paraformalde- 
hyde-glutaraldehyde fixative. The latter were postfixed in \ ( /r OsO 4 . dehydrated 

1 Contribution no. 211 from the Institute for Molecular and Cellular Evolution, University 
of Miami. Work was supported in part by NIH grant (5T1-HD-05 to 09) to the Fertilization 
and Gamete Physiology Training Program at the Marine Biological Laboratory. 

" XIH Career Development Awardee. 

358 



REPRODUCTION IN LIBIXIA 



359 




FIGURE 1. Ventral view of immature (A) and mature (B) female Libhiin. 
Note difference in outline of abdomen (arrows), X 1/2. 

and embedded in Araldite for electron microscopy. Paraffin sections (5 p) or thick 
plastic sections (1 /JL] were stained with toluidine blue to determine the extent of 
ovarian differentiation at various stages. 



OBSERVATIONS 



Se.viutl maturation and inoltiin/ 



Classification of females as immature or mature was determined on the basis of 
size and shape of their abdomens. The abdomen of immature females is narrow 
and does not extend to the base of the legs (Fig. 1 ). In the adult, it is rounded and 
almost reaches to the base of the legs (Fig. 1). Figure 2 indicates the carapace 
length in centimeters of immature and mature females. The maximum length of 
immature females seen from the wild populations was 6.0 cm. Mature females 



20 - 



10 - 






A 





6 



8 cm 



FIGURE 2. Carapace length of individual crabs. A indicates the length of 318 mature females, 

B the length of 202 immature females. 



360 



GERTRUDE W. HINSCH 



exceed 4.0 cm. Thus, in the 4-6 cm range, females may be either mature or im- 
mature. 

Among the immature spider crabs studied (above 1.9 cm carapace length), molt- 
ing normally was observed to begin in August or September. The time varied from 
middle August during the summer of 1968 to the middle of September in 1970. 
Successful molting occurred even when large numbers of crabs occupied the same 
aquarium. Carapace length of several immature control females prior to molt and 
length and sexual state after a normal molt are shown in Figure 3. Crabs under- 
going normal ecdysis vary in the amount of growth following the molt as well as the 
state of sexual maturation. No mature females were ever observed undergoing 
ecdysis. 

Immature and mature females were destalked and observed for indications of 
molting. None of the 43 mature destalked females molted or upon dissection 
showed signs of molting. Among the 51 immature females which were destalked 
in late July or August, most began molting within two weeks after the operation. 
Those destalked in May or June showed no signs of molting within two weeks. 
Molt, however, was initiated after four to five weeks. Destalked crabs had diffi- 
culty in completing the molting process and although the number of crabs per 
aquarium was reduced (10 per aquarium), a molting crab frequently was attacked 



10 - 

5 - 


5 - 





<o 5 - 



5 - 



- 1 



C 



D 




8 cm 



FIGURE 3. A-C, carapace length of intact females ; A carapace length of 76 immature 
control female crabs, B Thirty seven females which remained immature following a normal 
molt, C Twenty one females which molted to maturity, D-E Destalked immature females. 
In D, the carapace length (in centimeters) prior to the molt of 38 immature females is indi- 
cated. In E, the carapace length of 32 recovered females all of which retained the immature 
apron. 



REPRODUCTION IN LIBIM.l 



361 



and eaten by her aquarium mates. Such behavior was not seen in molting, normal, 
unoperated crabs even under crowded conditions (30-40 per aquarium). In addi- 
tion, destalked crabs frequently decorated their carapaces with bits of shell, sea 
urchin spines, and other debris found on the aquarium bottom. Unoperated crabs 
rarely did such in these experiments. 

As reported earlier (Hinsch, 1970), destalked immature females rarely molted 
to the mature state. This was particularly true when immature females of various 
sizes were destalked several weeks prior to the time when their controls commenced 
to molt naturally. Giant immature females were produced. As shown in Figure 3, 
one might have expected on the basis of carapace length alone (compare with Fig. 2) 
that several of these crabs would have been mature. However, immature females 
which were destalked and which molted at the same time as their controls fre- 
quently molted to maturity. Two destalked females who had molted once following 
the operation underwent an additional molt two weeks after the first. This molt was 
fatal. The abdomen and ovaries of these two crabs were those of an immature 
female. 

TAHU-; I 

Dcstalkuig experiments f unit it re female Libinia. The experimental females were 

destalked on August 7, I960 and observed periodically thereafter. 

Oviposition was stimulated in the operated females 





Egg mass/No, of crabs 




Aug. 


AUK. 


Aug. 


Sept. 


Sept. 


Sept. 


Sept. 




11 


18 


26 


2 


11 


15 


23 


Experimental* (destalked ) 
















20 with eggs in brood pouch 


19/19 


10/19 14/16 


14/15 


6/15 


9/15 


10/15 


16 without eggs in brood pouch 


0/14 


0/14 


11/12 


9/12 


5/12 


8/11 


9/9 


Controls 
















15 (eyestalks not removed) 


2/15 


3/14 


3/13 


1/14 


0/14 


0/14 


0/14 



Reproductive cycles 

The breeding behavior and reproductive cycles of the female Libinia have been 
described (Hinsch, 1968). Mature females apparently oviposit for the first time 
in late May or early June. This brood is often quite small. Tt is followed by 
additional spawnings at 25 days intervals. The time of last oviposition varies 
seasonally. 

To determine the role of the eyestalk hormones on reproduction, mature fe- 
males were destalked during various stages of their ovarian cycles. Following the 
operation, the crabs were placed in aquaria with some males and observed. The 
25 clay brood period was unaltered and the females continued to attract males at 
the time of zoeae hatching as did those in the control tanks (Hinsch, 1968). 

In July 1%8. 43 mature females with broods were destalked. On September 
23, 24 of the 27 survivors had orange egg masses (new). Among the wild 
population, onlv 3 of ''3 mature females carried broods at this time. 



362 



GERTRUDE W. HINSCH 



In early August 1969, mature females with broods and females who had been 
without broods for at least one week were subjected to destalking and observed 
periodically (Table I). 13 y late September, only females which had been destalked 
carried egg masses in their brood pouches. 

On September 3. 1970, these experiments were repeated on an additional 130 
crabs. Seventy females with broods, many of them relatively new (orange egg 
masses), were used as controls. Sixty females lacking broods were separated into 
two groups. Thirty were left unoperated and placed in an aquarium with males. 
The other thirty were destalked prior to being placed with males in another 
aquarium. All crabs were observed periodically and the presence or absence of a 
brood noted (Table II). The operated females were frequently observed mating 
with males, while unoperated females rarely mated at this time of the year. On 
September 26, 27 of the 28 surviving operated females contained orange (new) 
egg masses in their brood pouches. Of the 99 unoperated females, 5 had new 
(orange) egg masses, 5 had brown egg masses containing xoeae about to hatch and 
88 were without egg masses. As noted earlier (Hinsch. 1968) a female need not 
mate immediately before each brood of eggs is laid to produce viable young. 



TABLE II 

Presence or absence of egg masses in the brood pouches was observed in 

mature females. As indicated, oviposition was stimulated in the 

females which have been destalked 





Egg masses/No, of crab- 


(1970) 






Sept. 3 


Sept. 21 


Sept. 26 


Experimental females (destalked) 


0/30 


14/29 


27/28 


Control females (non-destalked) 








Females with broods 


70/70 


12/70 


10/70 


Females without broods 


0/30 


1/29 


1/29 



Ovarian development 

Dissection of immature females revealed ovaries which were small, white H- 
shaped organs. Sections of these ovaries showed large masses of immature oocytes 
near a central core of syncytial cells and showed no signs of vitellogenesis (Fig. 4). 
The ovaries of females which have just molted to maturity are also white and only 
slightly larger than those of immature females. These oocytes are small, have 
vacuolated nucleoli and no yolk. One month following the molt to maturity, many 
of the crabs have ovaries which are enlarged, orange in color and have oocytes in 
which vitellogenesis is well advanced. Following oviposition, ovarian development 
takes place in the ovary of the mature female as she is brooding her young. Eggs 
collected near the end of a brooding cycle have well developed yolk and a forming 
egg coat (Fig. 5). Females who are about to release zoeae during the breeding 
season have oocytes in their ovaries which are fully developed, surrounded by an 
egg envelope and lack follicle cells. As each brood hatches, a new mass of eggs 
is generally oviposited after approximately 6 to 12 hours (Hinsch, 1968). Ovaries 
of females at the end of the breeding season vary in stage of maturation, although 



REPRODUCTION IN LIBIN1A 



363 



jsp- *js* " * ' . ' . : ,:, jjgTfrj w' 

S?-' ^^ j&'~ ' ' ; - v ? P^' ^ ' ^j: " ^?" 

W&'.-'z 4&- : : - "'-^ fl?rf!4 



. 

. vQC. Ai r*E 







i^A 



'? ' - . L -aS*i'li.i. "'-' --vv's. -?& *^w i "SB 

P^|^:f ^f^^J 




"*, 







. 

-* ** * i t 





;v- ,., B ;.-. 

I I ^c 

" -:'- III '\ ; ; ix^ 

.- '' I/ ' ..' i -^x V *^" : v " ; " -. '"' v j< ,-";'" 





^;fv^ P?." ' : ^ 



^ " f -'.?f 
^ >-"'!. 

-*; ,?* 





r r ' " l '- ; -, 

FIGURE 4. Ovary from an immature female collected in mid-August. Premolt condition 
is unknown. Many small oocytes are apparent. 

FIGURE 5. Oocyte from mature female brooding young. The oocyte (O) is surrounded by 
forming egg coat (EC) and follicle cells (FC). 

FIGURE 6. Mature oocytes from bright orange ovary of female at the end of the breeding 
season. Follicle cells (FC) still surround the oocyte and egg coat (EC) has only partially 
formed. 

FIGURE 7. Oocyte from orange ovary of a mature female in mid-November. In this 
instance vitellogenesis including pinocytosis (arrow) is seen and beginnings of egg coat (EC) 
are seen between oocyte (O) and follicle cells (FC). Whether this female had recently molted 
to maturity is not known. 



364 GERTRUDE \V. HINSCH 

most are usually bright orange and contain large oocytes rilled with yolk (Figs. 
6 and 7). In many cases these oocytes are still surrounded by follicle cells and may 
or may not show traces of an egg envelope (Figs. 6 and 7). Thus, these oocytes 
apparently overwinter until the next breeding season commences the following 
spring and are released as the first brood of the year. Mature females shipped 
to Florida from Woods Hole during the winter months have well developed ovaries 
and some days after acclimatizing to the warmer water temperature oviposit. 

Seminal receptacles are absent in the immature females and very small in those 
newly molted to adulthood. In addition, newly molted adult females have not been 
observed mating in the soft condition. 

DISCUSSION 

Arthropod metamorphosis, particularly phases of molting and sexual matura- 
tion, have been extensively studied in recent years. It is particularly well docu- 
mented in the insects. Although crustaceans have been studied less extensively, 
many similarities to the insects have been noted. Ecdysis is controlled by neuro- 
section from the X-organ (crustaceans) of the eyestalk or protocerebrum (insects). 
This neurosecretion activates the Y-organ (crustaceans) or corpora cardiaca (in- 
sects) to produce a hormone which initiates molting. Molting can be initiated in 
crustaceans by the removal of the eyestalk with its contained X-organ-sinus gland 
complex (See Passano, 1960; Highnam and Hill, 1969; Adiyodi and Adiyodi, 
1970 for reviews). In Libinia the duration of time between eyestalk ablation and 
onset of molting varies and seems dependent on the relationship of time of ordi- 
nary ecdysis. Further factors may include temperature changes, nutrition, salinity 
and light-darkness periods as have been suggested by Aiken (1969) and Stephens 
(1952). Immature Libinia females molt following eyestalk ablation although ma- 
ture crabs never have been seen to molt after reaching maturity. Thus Libinia like 
Maja (Drach, 1939) apparently undergoes a terminal molt to maturity. Perhaps 
this may be true of all members of the family Maijdae. In Maja (Carlisle and 
Knowles, 1957) failure to molt beyond the terminal stage has been attributed to 
the degeneration of the Y-organ. Whether the Y-organ degenerates in Libinia is 
not known. 

Sex differentiation in crustaceans as in most animals is genetically determined 
although hormonally mediated. Sexual differentiation of the male is apparently con- 
trolled by the androgenic gland (Charniaux-Cotton. 1964; Charniaux-Cotton ct a!.. 
1966). Females develop in the absence of the androgenic substance secreted by 
this gland. They can be masculinized by transplantation of the androgenic gland 
to their bodies. In most crustaceans, the reproductive cycles and body growth 
cycles are closely related and regulated by interlinked control mechanisms. These 
include the eyestalk hormones which control molting, oogenesis, vitellogenesis and 
secretion of sex hormones (for review see Adiyodi and Adiyodi, 1970) in many 
crustaceans having preadult and multiple adult female instars. Reproductive hor- 
mones influence the synthesis and accumulation of a sex-related lipoprotein utilized 
in vitellogenesis (Adiyodi, 1968a. b). Such a lipoprotein is found in mature 
female Libinia undergoing vitellogenesis but is absent in the blood of immature 
females and males ( Bischoff and Telfer, University of Cincinnati and University of 
I Vnnsylvania. unpublished). 



REPRODUCTION IN LIBINIA 365 

In Libinia sexual maturation of the females apparently occurs only following 
the terminal molt. Reproduction in this form can thus be studied independently of 
molting and the factors controlling sexual maturation recognized. Evidence pre- 
sented here suggests some of the parameters of endocrine control of reproduction 
in Libinia. The eyestalk hormone apparently does not act to inhibit all stages of 
vitellogenesis. Evidence for this is the mature ovaries found in females out of the 
breeding season. In addition, the eyestalk hormone (s) seem to govern or regulate 
egg release. This is indicated by the continued oogenesis and oviposition beyond 
the normal season or resumption of oviposition following eyestalk ablation. The 
time delay between eyestalk removal and oviposition suggests that yet another 
factor which is controlled by the eyestalk hormone (s) may be involved. This 
could be a stimulating substance produced by the neurosecretory cells of, the cerebral 
and/or thoracic ganglia (Otsu, 1963; Parameswaran, 1955) or could simply be the 
time needed for metabolic breakdown of the inhibiting hormone following eyestalk 
ablation. The fact that destalked immature females do not molt to maturity also 
implies hormonal control from a source other than the X-organ-sinus gland com- 
plex. The exact nature and origin of such factor (s) is yet to be determined. In 
some crustaceans the Y-organ has been suggested as a source of a gonad-maturation 
factor. However, if in Libinia the Y-organ degenerates as in Maja following the 
molt to maturity, then one must rule out such a role and look elsewhere. 

Although the soft conditions following molting is generally considered favorable 
for mating in crustaceans it may be unimportant in Libinia since mating can occur 
in a hardened female (Hinsch, 1968). As reported here, newly molted mature 
females have not been observed to mate although such may occur under natural 
conditions. Males have not been seen carrying premolt females about as happens 
in other species, e.g., Portunus (Ryan, 1967), Maja (Schone, 1968). The small 
size of the seminal receptacle in the newly molted adult Libinia suggests that the 
initial mating may occur much later in the hardened female. Hartnoll (1963) has 
reported that female Maja and Pisa, species belonging to the same superfamily as 
Libinia, are physically incapable of mating prior to the molt to puberty. This is 
the terminal molt. In general, ovarian maturation in members of this superfamily 
(i.e., Pisa, Inachus, Hyas) occurs after the molt to maturity (Hartnoll, 1963) as 
we have found in Libinia. 

SUMMARY 

1. Carapace length is not sufficient for determining state of sexual maturation 
of female Libinia. Females in range of 4-6 cm carapace length may be mature or 
immature. Shape of the abdomen distinguishes between mature and immature 
females. 

2. Eyesalk ablation of mature female Libinia results in extended periods of or 
initiation of the ovigerous state but does not appear to initiate molting. Breeding 
behavior and reproductve cycles seem unaltered by destalking. 

3. Immature female Libinia which have had their eyestalks removed molt 
precociously but rarely to maturity. 

4. Libinia apparently undergo a terminal molt to maturity. Ovarian development 
and vitellogenesis occur only in mature females. 



366 GERTRUDE W. H1NSCH 

LITERATURE CITED 

ADIYODI, K. G., AND R. G. ADIYODI, 1970. Endocrine control of reproduction in decapod 

Crustacea. Biol. Rev., 45 : 121-165. 
ADIYODI, R. G., 1968a. Protein metabolism in relation to reproduction and molting in the crab 

Paratelphusa hydrodromous (Herbst.) Part I. Electrophoretic studies on the mode 

of utilization of soluble proteins during vitellogenesis. Indian J. Exp. Biol., 6: 144-147. 
ADIYODI, R. G., 1968b. Part II. Fate of conjugated proteins during vitellogenesis. Indian J. 

Exp. Biol, 6 : 200-203. 
AIKEN, D. E., 1969. Photoperiod, endocrinology and the crustacean molt cycle. Science, 164 : 

149-155. 
CARLISLE, D, B., AND F. G. KNOWLES, 1959. Endocrine Control in Crustaceans. Cambridge 

University Press, London and New York. 
CHARNIAUX-COTTON, H., 1964. Hormonal control of sex differentiation in invertebrates. Pages 

701-740 in R. L. DeHaan and H. Ursprung, Eds., Organo genesis. Holt, Rinehart and 

Winston, New York. 
CHARNIAUX-COTTON, H., C. ZERBIB AND J. J. MEUSY, 1966. Monographic de la glande 

androgene des Crustaces superieurs. Crustaccana, 10: 113-136. 
DRACH, P., 1939. Mue et cycle d'intermue chez les Crustaces Decapodes. Ann. Inst. Oceanog. 

(Paris), 19: 103-391. 
HARTNOLL, R. G., 1963. The biology of Manx spider crabs. Proc. Zool. Soc. London, 141 : 

423-496. 
HIGHNAM, K. C., AND L. HILL, 1969. The Comparative Endocrinology of the Invertebrates. 

American Elsevier, New York, 270 pp. 
HINSCH, G. W., 1968. Reproductive behavior in the spider crab, Libinia emarginata L. Biol. 

Bull., 135 : 273-278. 
HINSCH, G. W., 1970. Some factors controlling reproduction in the spider crab, Libinia 

emarginata. Biol. Bull, 139 : 410. 
KARNOVSKY, M. J., 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use 

in electron microscopy. /. Cell Biol, 27 : 137A. 
OTSU, T., 1963. Bihormonal control of sexual cycle in the freshwater crab, Potamon dehaani. 

Embryologia, 8 : 1-20. 
PARAMESWARAN, R., 1955. Neurosecretory cells in Paratelphusa hydrodromous (Herbst.). 

Current Sci., 24 : 23-24. 
PASSANO, L. M., 1960. Molting and its control. Pages 473-536 in T. Waterman, Ed., 

Crustacea. Vol. I. Academic Press, New York. 
RYAN, E. P., 1967. Structure and function of the reproductive system of the crab, Portunus 

sanguinolcntus (Herbst.) (Brachyura: Portunidae). II. The female system. Proc. 

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SCHONE, H., 1968. Agonistic and sexual display in aquatic and semiterrestrial brachyuran 

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Reference : Biol. Bull., 143 : 367-391. (October, 1972) 



SUGAR RELEASE AND PENETRATION IN INSECT FAT BODY : 

RELATIONS TO REGULATION OF HAEMOLYMPH 

TREHALOSE IN DEVELOPING STAGES OF 

HYALOPHORA CECROPIA 

ARTHUR M. JUNGREIS * AND G. R. WYATT 
Department of Biology, Yale University, Neiv Haven, Connecticut 06520 

In insects, the fat body is the chief site of Synthesis of the disaccharide treha- 
lose, which is generally the predominant circulating sugar (review: Wyatt, 1967). 
There is evidence for homeostatic regulation of haemolymph 'trehalose levels 
(Saito, 1963; Friedman, 1967; Wyatt, 1967; Nettles, Parro, Sharbaugh and 
Mangum, 1971) and, to account for this, feedback mechanisms have been proposed. 
While inhibition and activation of enzymes concerned with trehalose synthesis 
have been demonstrated in vitro (Murphy and Wyatt, 1965; Friedman, 1968), 
the mechanisms responsible for regulation in vivo are unclear. One hitherto un- 
answered question is how an insect can maintain its haemolymph sugar at distinctly 
different levels in its different developmental stages, as, for example, the cecropia 
silkmoth has been shown to do (Wyatt, 1967). 

The dynamic relations between intracellular and extracellular trehalose are 
clearly important for the regulation of trehalose synthesis, yet very little is known 
about these. Mochnacka and Petryszyn (1959) found trehalose to be higher in 
haemolymph than in "bled tissues" of pupae of the sphingid Celerio euphorbiae, 
and larval and adult tissues of this species apparently contained no trehalose. Fat 
body of cecropia silkmoth larvae (Wyatt, 1967, page 297) and wax moth larvae 
(Lenartowicz, Zaluska and Niemierko, 1967), after rinsing in saline solutions, 
was found to contain trehalose at levels much lower than those in the haemolymph 
of the respective insects. These reports suggest that movement of trehalose from 
fat body to haemolymph might require active transport, albeit active transport of 
sugars has never been demonstrated in an insect. In fact, the absorption of sugars 
from the insect gut, in contrast to analogous processes in vertebrates, depends upon 
simple diffusion (Wyatt, 1967). 

Fat body of larval blowflies (Phormia reyina), on the other hand, contained 
trehalose even though none could be found in the haemolymph of the same stage 
(Wimer, 1969). 

In view of these problems, we have examined the relations between fat body 
and haemolymph sugar in several insect species. We find that fat body analyzed 
without prior rinsing may contain sugar at levels lower than, equal to, or higher 
than those in the haemolymph, but during rinsing in isosmotic media much of the 
tissue sugar is rapidly released. During ontogeny of the cecropia silkmoth, the 
quantitative relationships between haemolymph and fat body trehalose change 
markedly. Correlated with these changes are alterations in fat body function with 

1 Present address : Department of Zoology, Ohio University, Athens, Ohio 45701. 

367 



368 A. M. JUNGREIS AND G. R. WYATT 

respect to release of endogenous and penetration of exogenous trehalose, which 
we interpret as reflecting changes in the cell membrane having regulatory signifi- 
cance. 

MATERIALS AND METHODS 
Animals and media 

Most of the experiments were conducted with cecropia silkmoths (Hyalophora 
cccropia), reared from genetically mixed stock either outdoors on wild cherry 
foliage or in the laboratory at 25 C on an artifical diet ( Riddiford, 1968) under 
16 hours light per day. After spinning their cocoons, outdoor reared animals 
were held at 25 C for 2 I- months and then (a) chilled at 6 C to activate the 
neuroendocrine system for development, or (1>) debrained to establish permanent 
diapause (Williams, 1946) and then kept at 15 C, or (c) left at 25 C. at which 
temperature diapause persisted in most individuals for several months. In many 
instances, pupal respiration was measured, and the criterion for diapause was an O^. 
consumption of less than 20 /xl per gram live weight per hour (Schneiderman and 
Williams, 1953). Pharate adults ("developing adults") were obtained by incubat- 
ing previously chilled pupae at 25 C, and stages were recognized as described by 
Schneiderman and Williams (1954). Insects of other species, used in a few 
experiments, were obtained as described in the footnotes to Table I. 

Injections were made through the thoracic tergum of pupae in volumes not 
more than 50 p\, and between the first pair of dorsal tubercles of larvae in not 
more than 100 /xl. Experimental injury consisted of making 20 punctures with a 
25 gauge needle in the thoracic tergum. which were then sealed with melted 
paraffin wax. Haemolymph \vas collected at the time of injury by puncturing a 
wing sac with the tip of a scalpel and then sealing the hole. When haemolymph 
was collected for use in media, a few crystals of phenylthiourea were added to pre- 
vent darkening due to the action of tyrosinase. 

The media used for rinsing and incubation of tissue from all insects except 
Blaberus discoidalis were: (i) that of Reddy and Wyatt (1967) (RW medium) 
containing NaCl 20 HIM, KC1 80 HIM, CaCU 4 HIM, MgQ 2 15 HIM, phosphate 
8 mM, and 20 amino acids (total 91 HIM), the sugars in the published medium 
being omitted, with pH 6.5 and osmotic pressure approximately 450 milliosmolar ; 
(ii) modified RW medium in which the concentrations of NaCl and KC1 were 
reduced to 4 and 50 HIM, respectively (total 360 milliosmolar) ; and (iii) high 
K+-Mg++ lepidopteran saline, modified after Weevers (1966) and Pan, Bell 
and Telfer (1969), which contained XaCl 4 HIM, KG 140 HIM, CaCU 4 HIM and 
MgCl2 15 HIM (total 350 milliosmolar). In initial experiments RW medium was 
used, while later the modified RW medium was substituted, and no differences 
were observed in the results. For B. discoidalis, a medium was used consisting 
of NaCl 130 mM. KC1 10.3 HIM, CaCl L . 4.5 HIM, MgClo 8 HIM. and phosphate 
buffer 5 mM, together with the amino acid mixture of RW medium (pH 6.5, 425 
milliosmolar; cf. Van Asperen and Van Esch, 1956). 

The sutjar content of haemolymph and fat body, and cfflit.v from fat body in vitro 

Haemolymph and fat body were taken from individual insects for analysis. 
The fat body was dissected out. blotted by drawing it repeatedly across Glassine 



SUGARS AND INSECT FAT BODY 369 

weighing paper (Eli Lilly and Company, Indianapolis, Indiana), and portions of 
50-500 mg were homogenized for analysis immediately, or after immediate freezing, 
or after incubation at 25 in not less than 1.8 ml of medium. For analysis, blotted 
tissue samples w r ere weighed to the nearest milligram on a torsion balance and 
ground in glass homogenizers in three volumes of 6.7% trichloracetic acid. Homo- 
genates